Methods for treating hyperhidrosis

ABSTRACT

Methods for treating hyperhidrosis is disclosed herein. In one embodiment, the method includes a step of administering a neurotoxin to a skin area to alleviate excessive sweating. In another embodiment, the method employs a needleless injector to affect the administration of a neurotoxin, for example botulinum toxin type A.

BACKGROUND

[0001] In mammals, for example human beings, sweating is a normalthermoregulation process. Also, sweating is a normal physiologicalresponse to a psychological stress or emotional stimuli. For mostpeople, sweating is only a minor cosmetic annoyance. For others,however, sweating may be excessive and become a socially or medicallycrippling handicap. The present invention relates to methods fortreating excessive sweating in a mammal, including a human being,wherein the methods include a step of administering a neurotoxin to amammal.

[0002] Hyperhidrosis is a disorder in which there is an exaggeratedsweat secretion involving both the eccrine and the apocrine sweatglands. The excessive sweating usually occurs in the palms, soles, andaxillae. Palmar hyperhidrosis is a condition of excessive sweating inthe hand. Such condition may be socially embarrassing. Plantarhyperhidrosis is a condition of excessive sweating in the foot. Thiscondition may cause blisters, infections, and bromohidrosis. Axillaryhyperhidrosis is a condition of excessive sweating in the armpit. Inaxillary hyperhidrosis, as much as 26 mL/h of sweat can be excreted fromeach armpit. Such excessive sweating is not only socially embarrassingbut may even cause staining and rotting of clothes.

[0003] Presently, the cause of hyperhidrosis is unknown. However, whatis known is that the 3 to 4 million sweat glands of the body are underthe control of the hypothalamus and the sympathetic system. Afferentimpulses from sensors on the skin and other parts of the body travel tothe hypothalamus, which integrates the information for chemoregulationof the body. The preoptic area of the anterior hypothalamus then sendsefferent impulses via sympathetic fibers back out to the body. SegmentT2 to T4 of the spinal chord innervate the head and neck area; fibers insegment T2 to T8 innervate the upper limbs; fibers in segment T6 to T10innervate the trunk; and finally fibers in T11 to T12 innervate thelower extremities.

[0004] Although sympathetic innervations typically rely on adrenergicneurotransmitters, acetylcholine is the neurotransmitter released by thesympathetic nerve terminals involved in innervating the sweat glands.However, that is not to say that only acetylcholines can innervate thesweat glands. Some reports have shown that eccrine and apocrine glandsrespond to α- and β-adrenergic agonists as well.

[0005] Although the hypothalamus has a significant role in controllingthe rate of sweating, other physical variables may affect the rate ofsweat secretion. For example, sweating rate may also be affected byvariables such as wetness and blood flow. Additionally, the rate ofsweating varies greatly among people and is related to acclimatization,sex, age, and maybe even diet.

[0006] With respect to treating hyperhidrosis, various treatments arebeing used. For example, topical administration aluminum chloride is acommon practice. It is thought that aluminum chloride mechanicallyobstruct eccrine sweat glands to reduce sweating, although some evidenceshows that the reduction in sweat may result from atrophy of thesecretory cells. A downside of using aluminum chloride is that thealuminum chloride may react with the water content of the sweat to formhydrochloric acid. The formation of hydrochloric acid may cause severeskin irritation.

[0007] Other topical preparations are also being used. For example,treatment of plantar and palmar hyperhidrosis includes use ofglutaraldehyde and tannic acid (strong tea). However, this treatment maycause a browning of the skin.

[0008] Anticholinergics, both systemic and topical, are also being used.However, most patients cannot tolerate the side effects.

[0009] In addition to the described adverse effect of the above methods,the above treatment methods are effective to alleviate excessivesweating for only a brief duration of time, thus requiring frequenttreatments, i.e. daily or weekly.

[0010] Surgical treatment involving sweat gland excission andsympathectomy may provide for a longer duration of alleviation fromhyperhidrosis. However, these invasive treatments are rarely indicateddue to the adverse consequences and cost. For example, surgery may causecontractures. Sympathectomy may result in complications includinginfection, pneumothorax, Horner's syndrome, resumption of sweating, andcompensatory hyperhidrosis. Additionally, hyperhidrosis may resume aftersurgery or sympathectomy.

[0011] Botulinum Toxin

[0012] The anaerobic, gram positive bacterium Clostridium botulinumproduces a potent polypeptide neurotoxin, botulinum toxin, which causesa neuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

[0013] BoNT/A is the most lethal natural biological agent known to man.About 50 picograms of botulinum toxin (purified neurotoxin complex)serotype A is a LD₅₀ in mice. One unit (U) of botulinum toxin is definedas the LD₅₀ upon intraperitoneal injection into female Swiss Webstermice weighing 18-20 grams each. Seven immunologically distinct botulinumneurotoxins have been characterized, these being respectively botulinumneurotoxin serotypes A, B, C₁, D, E, F and G each of which isdistinguished by neutralization with serotype-specific antibodies. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke. For example, it has been determined that BoNt/A is 500 times morepotent, as measured by the rate of paralysis produced in the rat, thanis botulinum toxin serotype B (BoNT/B). Additionally, BoNt/B has beendetermined to be non-toxic in primates at a dose of 480 U/kg which isabout 12 times the primate LD₅₀ for BoNt/A. Botulinum toxin apparentlybinds with high affinity to cholinergic motor neurons, is translocatedinto the neuron and blocks the release of acetylcholine.

[0014] Botulinum toxins have been used in clinical settings for thetreatment of neuromuscular disorders characterized by hyperactiveskeletal muscles. BoNt/A has been approved by the U.S. Food and DrugAdministration for the treatment of blepharospasm, strabismus andhemifacial spasm. Non-serotype A botulinum toxin serotypes apparentlyhave a lower potency and/or a shorter duration of activity as comparedto BoNt/A. Clinical effects of peripheral intramuscular BoNt/A areusually seen within one week of injection. The typical duration ofsymptomatic relief from a single intramuscular injection of BoNt/Aaverages about three months.

[0015] Although all the botulinum toxins serotypes apparently inhibitrelease of the neurotransmitter acetylcholine at the neuromuscularjunction, they do so by affecting different neurosecretory proteinsand/or cleaving these proteins at different sites. For example,botulinum serotypes A and E both cleave the 25 kiloDalton (kD)synaptosomal associated protein (SNAP-25), but they target differentamino acid sequences within this protein. BoNT/B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin serotype C₁ (BoNT/C₁) has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes.

[0016] Regardless of serotype, the molecular mechanism of toxinintoxication appears to be similar and to involve at least three stepsor stages. In the first step of the process, the toxin binds to thepresynaptic membrane of the target neuron through a specific interactionbetween the H chain and a cell surface receptor; the receptor is thoughtto be different for each serotype of botulinum toxin and for tetanustoxin. The carboxyl end segment of the H chain, H_(c), appears to beimportant for targeting of the toxin to the cell surface.

[0017] In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

[0018] The last step of the mechanism of botulinum toxin activityappears to involve reduction of the disulfide bond joining the H and Lchain. The entire toxic activity of botulinum and tetanus toxins iscontained in the L chain of the holotoxin; the L chain is a zinc (Zn++)endopeptidase which selectively cleaves proteins essential forrecognition and docking of neurotransmitter-containing vesicles with thecytoplasmic surface of the plasma membrane, and fusion of the vesicleswith the plasma membrane. Tetanus neurotoxin, botulinum toxin/B/D,/F,and/G cause degradation of synaptobrevin (also called vesicle-associatedmembrane protein (VAMP)), a synaptosomal membrane protein. Most of theVAMP present at the cytosolic surface of the synaptic vesicle is removedas a result of any one of these cleavage events. Each toxin specificallycleaves a different bond.

[0019] The molecular weight of the botulinum toxin protein molecule, forall seven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the BoNt/Acomplex can be produced by Clostridial bacterium as 900 kD, 500 kD and300 kD forms. BoNT/B and C₁ are apparently produced as only a 500 kDcomplex. BoNT/D is produced as both 300 kD and 500 kD complexes.Finally, BoNT/E and F are produced as only approximately 300 kDcomplexes. The complexes (i.e. molecular weight greater than about 150kD) are believed to contain a non-toxin hemaglutinin protein and anon-toxin and non-toxic nonhemaglutinin protein. These two non-toxinproteins (which along with the botulinum toxin molecule comprise therelevant neurotoxin complex) may act to provide stability againstdenaturation to the botulinum toxin molecule and protection againstdigestive acids when toxin is ingested. Additionally, it is possiblethat the larger (greater than about 150 kD molecular weight) botulinumtoxin complexes may result in a slower rate of diffusion of thebotulinum toxin away from a site of intramuscular injection of abotulinum toxin complex.

[0020] In vitro studies have indicated that botulinum toxin inhibitspotassium cation induced release of both acetylcholine andnorepinephrine from primary cell cultures of brainstem tissue.Additionally, it has been reported that botulinum toxin inhibits theevoked release of both glycine and glutamate in primary cultures ofspinal cord neurons and that in brain synaptosome preparations botulinumtoxin inhibits the release of each of the neurotransmittersacetylcholine, dopamine, norepinephrine, CGRP and glutamate.

[0021] BoNt/A can be obtained by establishing and growing cultures ofClostridium botulinum in a fermenter and then harvesting and purifyingthe fermented mixture in accordance with known procedures. All thebotulinum toxin serotypes are initially synthesized as inactive singlechain proteins which must be cleaved or nicked by proteases to becomeneuroactive. The bacterial strains that make botulinum toxin serotypes Aand G possess endogenous proteases and serotypes A and G can thereforebe recovered from bacterial cultures in predominantly their active form.In contrast, botulinum toxin serotypes C₁, D and E are synthesized bynonproteolytic strains and are therefore typically unactivated whenrecovered from culture. Serotypes B and F are produced by bothproteolytic and nonproteolytic strains and therefore can be recovered ineither the active or inactive form. However, even the proteolyticstrains that produce, for example, the BoNt/B serotype only cleave aportion of the toxin produced. The exact proportion of nicked tounnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the BoNt/B toxin is likely to be inactive,possibly accounting for the known significantly lower potency of BoNt/Bas compared to BoNt/A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that BoNt/B has, upon intramuscular injection,a shorter duration of activity and is also less potent than BoNt/A atthe same dose level.

[0022] It has been reported that BoNt/A has been used in clinicalsettings as follows:

[0023] (1) about 75-125 units of BOTOX®¹ per intramuscular injection(multiple muscles) to treat cervical dystonia;

[0024] (2) 5-10 units of BOTOX® per intramuscular injection to treatglabellar lines (brow furrows) (5 units injected intramuscularly intothe procerus muscle and 10 units injected intramuscularly into eachcorrugator supercilii muscle);

[0025] (3) about 30-80 units of BOTOX® to treat constipation byintrasphincter injection of the puborectalis muscle;

[0026] (4) about 1-5 units per muscle of intramuscularly injected BOTOX®to treat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

[0027] (5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

[0028] (6) to treat upper limb spasticity following stroke byintramuscular injections of BOTOX® into five different upper limb flexormuscles, as follows:

[0029] (a) flexor digitorum profundus: 7.5 U to 30 U

[0030] (b) flexor digitorum sublimus: 7.5 U to 30 U

[0031] (c) flexor carpi ulnaris: 10 U to 40 U

[0032] (d) flexor carpi radialis: 15 U to 60 U

[0033] (e) biceps brachii: 50 U to 200 U. Each of the five indicatedmuscles has been injected at the same treatment session, so that thepatient receives from 90 U to 360 U of upper limb flexor muscle BOTOX®by intramuscular injection at each treatment session.

[0034] The success of BoNt/A to treat a variety of clinical conditionshas led to interest in other botulinum toxin serotypes. A study of twocommercially available BoNT/A preparations (BOTOX® and Dysport®) andpreparations of BoNT/B and F (both obtained from Wako Chemicals, Japan)has been carried out to determine local muscle weakening efficacy,safety and antigenic potential. Botulinum toxin preparations wereinjected into the head of the right gastrocnemius muscle (0.5 to 200.0units/kg) and muscle weakness was assessed using the mouse digitabduction scoring assay (DAS). ED₅₀ values were calculated from doseresponse curves. Additional mice were given intramuscular injections todetermine LD₅₀ doses. The therapeutic index was calculated as LD₅₀/ED₅₀.Separate groups of mice received hind limb injections of BOTOX® (5.0 to10.0 units/kg) or BoNt/B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg for BoNt/Bor 0.15 ng/kg for BOTOX®). Peak muscle weakness and duration were doserelated for all serotypes. DAS ED₅₀ values (units/kg) were as follows:BOTOX®: 6.7, Dysport®: 24.7, BoNt/B: 27.0 to 244.0, BoNT/F: 4.3. BOTOX®had a longer duration of action than BoNt/B or BoNt/F. Therapeutic indexvalues were as follows: BOTOX®: 10.5, Dysport®: 6.3, BoNt/B: 3.2. Waterconsumption was greater in mice injected with BoNt/B than with BOTOX®,although BoNt/B was less effective at weakening muscles. After fourmonths of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of 4(where treated with 6.5 ng/kg) rabbits developed antibodies againstBoNt/B. In a separate study, 0 of 9 BOTOX® treated rabbits demonstratedantibodies against BoNt/A. DAS results indicate relative peak potenciesof BoNt/A being equal to BoNt/F, and BoNt/F being greater than BoNt/B.With regard to duration of effect, BoNt/A was greater than BoNt/B, andBoNt/B duration of effect was greater than BoNt/F. As shown by thetherapeutic index values, the two commercial preparations of BoNt/A(BOTOX® and Dysport®) are different. The increased water consumptionbehavior observed following hind limb injection of BoNt/B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to BoNt/A, it is necessary to increase doses of theother serotypes examined. Increased dosage can comprise safety.Furthermore, in rabbits, serotype B was more antigenic than was BOTOX®,possibly because of the higher protein load injected to achieve aneffective dose of BoNt/B.

[0035] The tetanus neurotoxin acts mainly in the central nervous system,while botulinum neurotoxin acts at the neuromuscular junction; both actby inhibiting acetylcholine release from the axon of the affected neuroninto the synapse, resulting in paralysis. The effect of intoxication onthe affected neuron is long-lasting and until recently has been thoughtto be irreversible. The tetanus neurotoxin is known to exist in oneimmunologically distinct serotype.

[0036] Acetylcholine

[0037] Typically only a single type of small molecule neurotransmitteris released by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic and most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition of theheart by the vagal nerve.

[0038] The efferent signals of the autonomic nervous system aretransmitted to the body through either the sympathetic nervous system orthe parasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

[0039] Acetylcholine activates two types of receptors, muscarinic andnicotinic receptors. The muscarinic receptors are found in all effectorcells stimulated by the postganglionic neurons of the parasympatheticnervous system, as well as in those stimulated by the postganglioniccholinergic neurons of the sympathetic nervous system. The nicotinicreceptors are found in the synapses between the preganglionic andpostganglionic neurons of both the sympathetic and parasympathetic. Thenicotinic receptors are also present in many membranes of skeletalmuscle fibers at the neuromuscular junction.

[0040] Acetylcholine is released from cholinergic neurons when small,clear, intracellular vesicles fuse with the presynaptic neuronal cellmembrane. A wide variety of non-neuronal secretory cells, such as,adrenal medulla (as well as the PC12 cell line) and pancreatic isletcells release catecholamines and insulin, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

[0041] A neuromuscular junction is formed in skeletal muscle by theproximity of axons to muscle cells. A signal transmitted through thenervous system results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscular junction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

[0042] Botulinum toxin has been shown to be effective in treatinghyperhidrosis. Especially, noteworthy is that botulinum toxin mayalleviate hyperhidrosis for up to 11 months. For example, Odderson,Dermatol Surg (1988) 24:1237-1241, discloses that intracutaneousinjections of botulinum toxin type A to the sweating area of the skinreduces excessive sweating; and Bushara et al., Clinical andExperimental Dermatology (1996) 21:276-278, disclose that subcutaneousinjections of botulinum toxin type A can selectively denervate the localsweat glands to produce an anhidrotic patch. The alleviation fromhyperhidrosis is up to 11 months.

[0043] The present methods for treating hyperhidrosis, for example thosedescribed by Odderson and Bushara et al., rely on the use of a needle,for example a 32 gauge needle, and a syringe for administration of adrug. Although the method of treatment is quite effective, this mode inadministration often causes pain. Additionally, it is very difficult todeliver the right amount of drug to the right layer of the skin, forexample the dermis layer. Consequently, an administration by a needlemay result in non-specific delivery, which lessens the treatmentefficacy. For example, misdirected injection into the epidermis orsubcutaneous tissue is not efficacious. Furthermore, non-specificdelivery may also causes adverse effects. For example, injection intothe subcutaneous tissue may result in diffusion of the toxin intosurrounding tissues. This non-specific diffusion of toxin may result inunwanted blockade of neuromuscular transmissions. Such neuromusculartransmission blockade may result in unwanted temporary loss of muscularfunctions, for example the hands. There remains a need for a method totreat hyperhidrosis more effectively.

SUMMARY

[0044] The methods of treating hyperhidrosis as described hereinovercome at least one of the aforementioned problems.

[0045] In accordance with the invention, a method for treatinghyperhidrosis in a mammal, for example a human being, includes the stepof locally administering a drug particle to an affected skin areawithout using a needle. For example, a needleless injector may be usedto affect a needleless administration or injection of the drug particle.For example, a needleless injector such as that sold by PowderJect,Oxford, UK, may be employed in accordance with this invention.

[0046] Further in accordance with the invention, the drug particle isadministered to at least one layer of the skin, for example theepidermis layer, the dermis layer and/or the hypodermis layer. Thedermis layer is believed to contain sweat glands and/or nervesinnervating the sweat glands.

[0047] Still further in accordance with the invention, the drug particleis administered to at least one layer of the skin without substantiallybeing administered to the muscle tissue beneath. The selectiveadministration may be affected through the use of a needleless injector.

[0048] Still further in accordance with the invention, the drug particlecomprises a neurotoxin. The neurotoxin may include a targetingcomponent, a therapeutic component and a translocation component. Thetargeting component may bind to a presynaptic nerve terminal, forexample a presynaptic nerve terminal of a cholinergic neuron. Forexample, the targeting component may include a carboxyl end segment of aheavy chain of a butyricum toxin, a tetani toxin, a botulimum toxin typeA, B, C₁, D, E, F, G or a variant thereof. The therapeutic component maysubstantially interfere with the release of neurotransmitters from aneuron or its terminals. For example, the therapeutic component mayinclude a light chain of a butyricum toxin, a tetani toxin, a botulimumtoxin type A, B, C₁, D, E, F, G or a variant thereof. The translocationcomponent may facilitate the transfer of at least a part of theneurotoxin into the cytoplasm of the target cell. For example, thetranslocation component may include an amino end fragment of a heavychain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B,C₁, D, E, F, G or a variant thereof.

[0049] Still further in accordance with the invention, the neurotoxin isbotulinum toxin type A. The neurotoxin may be produced recombinantly.For example, botulinum toxin type A may be produced recombinantly.

[0050] Still further in accordance with the invention, the drug particlemay include a neurotoxin and a carrier, wherein the neurotoxin is coatedonto the carrier. Any biologically compatible dense material such asgold, platinum and/or ice crystal may be used as a carrier. For example,a gold particle may be coated with a botulinum toxin type A to form adrug particle useful in this invention.

[0051] Each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present invention provided that the features included insuch a combination are not mutually inconsistent.

[0052] Definitions

[0053] Before proceeding to describe the present invention, thefollowing definitions are provided and apply herein.

[0054] “Affected skin area” means an area with excessive sweating.

[0055] “Drug particle” means a drug, for example, a neurotoxin, alone orin combination with other matters, for example, gold.

[0056] “Without using a needle” means injecting a measurable amount ofdrug particle without the use of a standard needle.

[0057] “Heavy chain” means the heavy chain of a clostridial neurotoxin.It preferably has a molecular weight of about 100 kDa and may bereferred to herein as H chain or as H.

[0058] “H_(N)” means a fragment (preferably having a molecular weight ofabout 50 kDa) derived from the H chain of a Clostridial neurotoxin whichis approximately equivalent to the amino terminal segment of the Hchain, or the portion corresponding to that fragment in the intact inthe H chain. It is believed to contain the portion of the natural orwild type clostridial neurotoxin involved in the translocation of the Lchain across an intracellular endosomal membrane.

[0059] “H_(C)” means a fragment (about 50 kDa) derived from the H chainof a clostridial neurotoxin which is approximately equivalent to thecarboxyl terminal segment of the H chain, or the portion correspondingto that fragment in the intact H chain. It is believed to be immunogenicand to contain the portion of the natural or wild type Clostridialneurotoxin involved in high affinity, presynaptic binding to motorneurons.

[0060] “Light chain” means the light chain of a clostridial neurotoxin.It preferably has a molecular weight of about 50 kDa, and can bereferred to as L chain, L or as the proteolytic domain (amino acidsequence) of a clostridial neurotoxin. The light chain is believed to beeffective as an inhibitor of neurotransmitter release when it isreleased into a cytoplasm of a target cell.

[0061] “Neurotoxin” means a chemical entity that is capable ofinterfering with the functions of a neuron. The “neurotoxin” may benaturally occurring or man-made.

[0062] “Variant” means a chemical entity which is slightly differentfrom a parent chemical entity but which still has a biological effect.The biological effect of the variant may be substantially the same orbetter than that of the parent. For example, a variant light chain of abotulinum toxin having at least one amino acid replaced, modified,deleted or added, may have the same or better ability to prevent therelease of neurotransmitter vesicles. Additionally, the biologicaleffect of a variant may be decreased. For example, a variant light chainof a botulinum toxin type A having a leucine base motif removed may havea shorter biological persistence than that of the parent (or native)botulinum toxin type A light chain.

DETAILED DESCRIPTION OF THE INVENTION

[0063] Methods for treating hyperhidrosis in mammals, for example humanbeings, are disclosed herein. In one broad embodiment, a method oftreating hyperhidrosis includes a step of locally administering a drugparticle to an affected skin area without using a needle, wherein thedrug particle is at least effective to reduce excessive sweating.

[0064] The skin has two distinct layers and varies in thickness fromabout 1.5 to about 4 mm or more, depending on the regions of the body.The first layer is the superficial layer called the epidermis. It is arelatively thick epithelium. Deep to the epidermis is the second layercalled the dermis. The dermis is a fibrous connective tissue andcomprises sweat glands and nerves, or nerve terminals, innervating suchsweat glands.

[0065] Just deep to the skin lies a fatty layer called the hypodermis,which may also be considered a part of a subcutaneous layer. Beneath thehypodermis or subcutaneous layer lies the deep fascial investment of thespecialized structures of the body, for example the muscles.

[0066] Accordingly, the method of this invention delivers the drugparticle to at least one layer of the skin. Preferably, the drug isdelivered to the layer of the skin in which the sweat glands and/or thenerve terminals innervating such sweat glands are found. For example, ina prefer embodiment, the drug particle is administered to the dermislayer. More preferably, the drug particle is administered to at leastone layer of the skin and not substantially to any tissues beneath theskin. For example, in one embodiment, the administered drug particle isdelivered to the dermis layer of the skin and not to the subcutaneouslayer. In a preferred embodiment, the administered drug particle isdelivered to the dermis layer of the skin and not to the muscle tissuesbeneath.

[0067] The administration of drug particles according to the inventionmay be affected through the use of a needleless injector, which is knownin the art. For example, Bellhouse et al. in U.S. Pat. Nos. 6,053,889('889), 6,013,050 ('050), 6,010,478 ('478), 6,004,286 ('286) and5,899,880 ('880) disclose novel needleless injectors. The disclosurestherein are incorporated in their entirety by reference herein. In oneembodiment, the needleless injector comprise an elongated tubular nozzleand is connected to or capable of connection to a suitable energizingmeans for producing a supersonic gas flow, for example a burst ofhelium, which accelerates fine drug particles to high velocity toward askin surface and into the skin surface. Such a device may be purchasedfrom PowderJect Pharmaceuticals, Oxford, UK. In a preferred embodiment,the gas pressure provided must be sufficient to discharge the drugparticles into a targeted site, for example the dermis, but not so greatas to damage the target. In another embodiment, the gas pressureprovided is sufficient to deliver the drug particle to a target site,for example the dermis, but not so great as to damage the skin surface,for example the epithelium. In a more preferred embodiment, the gaspressure is sufficient to deliver the drug particle to the dermis layer,but not to the layers below, for example the subcutaneous layer and/orthe muscle tissues. In an even more preferred embodiment, the gaspressure provided must be sufficient to discharge the drug particlesinto a targeted site, for example the dermis, but not so great as to (1)damage the skin surface and (2) deliver the drug particle to the muscletissue.

[0068] Advantages for using a needleless injector for the treatment ofhyperhidrosis according to the present invention include, for example,an optimal delivery to a specific skin layer, for example the dermislayer. Furthermore, in the case where the drug particle is delivered tothe dermis and not the muscle tissues, the treatment does not cause aloss of motor function in the area being treated. Also, the use of aneedleless injector according to the present invention improves clinicalsafety by eliminating the risk of infection from accidental injury withneedles or from potential splash back of bodily fluids from liquid jetinjectors, thereby avoiding the possibilities of cross-contamination ofblood-borne pathogens such as HIV and hepatitis B. The needlelessinjector, such as the PowderJect System, also offers an optimal andspecific delivery of drug particles to treat hyperhidrosis with no painor skin damage such as bruising or bleeding.

[0069] In another broad embodiment, the drug particle comprises aneurotoxin. The neurotoxin may include a targeting component, atherapeutic component and a translocation component. The targetingcomponent may bind to a presynaptic nerve terminal, for example apresynaptic nerve terminal of a cholinergic neuron. For example, thetargeting component may include a carboxyl end segment of a heavy chainof a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁,D, E, F, G or a variant thereof. In a preferred embodiment, thetargeting component comprises a carboxyl end segment of a heavy chain ofa botulinum toxin type A.

[0070] The therapeutic component may substantially interfere with therelease of neurotransmitters from a neuron or its terminals. Forexample, the therapeutic component may include a light chain of abutyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E,F, G or a variant thereof. In a preferred embodiment, the therapeuticcomponent comprises a light chain of a botulinum toxin type A.

[0071] The translocation component may facilitate the transfer of atleast a part of the neurotoxin into the cytoplasm of the target cell.For example, the translocation component may include an amino endfragment of a heavy chain of a butyricum toxin, a tetani toxin, abotulimum toxin type A, B, C₁, D, E, F, G or a variant thereof. In apreferred embodiment, the translocation component comprises an amino endfragment of a heavy chain of a botulinum toxin type A.

[0072] In one embodiment, the targeting component comprises a carboxylend fragment of a heavy chain of a botulinum toxin type A, thetherapeutic component comprises a light chain of a botulinum toxin typeA and the translocation component comprises an amine end fragment of aheavy chain of a botulinum toxin type A. In a preferred embodiment, theneurotoxin of the present invention comprises a botulinum toxin type A.For example, very useful botulinum toxin type A may be obtained fromAllergan, Inc., under the trade name BOTOX®.

[0073] In another broad aspect of this invention, recombinant techniquesare used to produce at least one of the components of the neurotoxins.The technique includes steps of obtaining genetic materials from eitherDNA cloned from natural sources, or synthetic oligonucleotide sequences,which have codes for one of the components, for example the therapeutic,translocation and/or targeting component(s). The genetic constructs areincorporated into host cells for amplification by first fusing thegenetic constructs with a cloning vectors, such as phages or plasmids.Then the cloning vectors are inserted into hosts, preferably E. coli's.Following the expressions of the recombinant genes in host cells, theresultant proteins can be isolated using conventional techniques. Theprotein expressed may comprise all three components of the neurotoxin.For example, the protein expressed may include a light chain ofbotulinum toxin type E (the therapeutic component), a heavy chain,preferably the H_(N), of a botulinum toxin type B (the translocationcomponent), and an H_(c) of botulinum toxin type A, which selectivelybinds to the motor neurons. In one embodiment, the protein expressed mayinclude less than all three components of the neurotoxin. In such case,the components may be chemically joined using techniques known in theart.

[0074] There are many advantages to producing these neurotoxinsrecombinantly. For example, production of neurotoxin from anaerobicClostridium cultures is a cumbersome and time-consuming processincluding a multi-step purification protocol involving several proteinprecipitation steps and either prolonged and repeated crystallization ofthe toxin or several stages of column chromatography. Significantly, thehigh toxicity of the product dictates that the procedure must beperformed under strict containment (BL-3). During the fermentationprocess, the folded single-chain neurotoxins are activated by endogenousClostridial proteases through a process termed nicking. This involvesthe removal of approximately 10 amino acid residues from thesingle-chain to create the dichain form in which the two chains remaincovalently linked through the intrachain disulfide bond.

[0075] The nicked neurotoxin is much more active than the unnicked form.The amount and precise location of nicking varies with the serotypes ofthe bacteria producing the toxin. The differences in single-chainneurotoxin activation and, hence, the yield of nicked toxin, are due tovariations in the type and amounts of proteolytic activity produced by agiven strain. For example, greater than 99% of Clostridial botulinumtype A single-chain neurotoxin is activated by the Hall A Clostridialbotulinum strain, whereas type B and E strains produce toxins with loweramounts of activation (0 to 75% depending upon the fermentation time).Thus, the high toxicity of the mature neurotoxin plays a major part inthe commercial manufacture of neurotoxins as therapeutic neurotoxins.

[0076] The degree of activation of engineered Clostridial toxins is,therefore, an important consideration for manufacture of thesematerials. It would be a major advantage if neurotoxins such asbotulinum toxin and tetanus toxin could be expressed, recombinantly, inhigh yield in rapidly-growing bacteria (such as heterologous E. colicells) as relatively non-toxic single-chains (or single chains havingreduced toxic activity) which are safe, easy to isolate and simple toconvert to the fully-active form.

[0077] With safety being a prime concern, previous work has concentratedon the expression in E.coli and purification of individual H and Lchains of tetanus and botulinum toxins; these isolated chains are, bythemselves, non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994);Zhou et al., Biochemistry 34:15175-15181 (1995), hereby incorporated byreference herein. Following the separate production of these peptidechains and under strictly controlled conditions the H and L subunits canbe combined by oxidative disulphide linkage to form the neuroparalyticdi-chains.

[0078] In one embodiment, the neurotoxin injected may be a nucleotidesequence. For example, the nucleotide sequence may be that of botulinumtoxin type A (SEQ. ID. #1), type B (SEQ. ID. #2 and #3), type C₁ (SEQ.ID #4), type D (SEQ. ID. #5), type E (SEQ. ID. #6 and #7), type F (SEQ.ID. #8) and type G (SEQ. ID. #9), variants thereof or fragments thereof.Preferably, the nucleotide fragment injected can encode a therapeuticcomponent, for example, a light chain.

[0079] A drug particle may comprise a neurotoxin alone or a neurotoxinin combination with other drugs and/or agents. In either case, theneurotoxin may be prepared as pharmaceutical compositions. Thecomposition may contain one or more added materials such as carriersand/or excipients. As used herein, “carriers” and “excipients” generallyrefer to substantially inert, non-toxic materials that do notdeleteriously interact with other components of the composition. Thesematerials may be used to increase the amount of solids in particulatepharmaceutical compositions, such as to form a powder of drug particlessuitable for use with a needleless injector. Examples of suitablecarriers include water, silicone, gelatin, waxes, and the like. Althougha naked nucleotide sequence may be injected in accordance with thisinvention, it is preferable that the injected nucleotide be accompaniedby a carrier, for example See Felgner et al, U.S. Pat. No. 5,459,127,the disclosure of which is incorporated in its entirety herein byreference.

[0080] Other suitable carriers include any high density, biologicallyinert materials. For example, tungsten, platinum, iridium gold and/orice crystal may be employed as carriers. In a preferred embodiment, thecarrier is less than about 10 μm, more preferably less than about 5 μm,even more preferably less than about 3 μm. High density carriers of suchsize may readily enter living cells without unduly injuring such cells.In one embodiment, a drug particle comprises a neurotoxin, for examplebotulinum toxin type A, and a carrier, for example a high densitymaterial of less than 5 μm, wherein the neurotoxin is coated onto thehigh density carrier using techniques commonly known in the art. Icecrystals and gold are preferred carriers of this invention. Ice crystalparticles are readily available in average sizes of 0.5 to 2.0 μm indiameter and are thus suited for intracellular delivery. Gold is also apreferred carrier, since gold has a high density and is relatively inertto biological materials and resists oxidation. Moreover, gold is readilyavailable in the form of spheres having an average diameter of fromabout 0.2 to about 3 μm. In a preferred embodiment, neurotoxin is coatedonto ice crystal and/or gold carriers to form drug particles. In a morepreferred embodiment, botulinum toxin type A is coated onto ice crystaland/or gold carriers to form drug particles to be used in accordancewith this invention.

[0081] Examples of normally employed “excipients,” includepharmaceutical grades of mannitol, sorbitol, inositol, dextrose,sucrose, lactose, trehalose, dextran, starch, cellulose, sodium orcalcium phosphates, calcium sulfate, citric acid, tartaric acid,glycine, high molecular weight polyethylene glycols (PEG), and the likeand combinations thereof. In one embodiment, the excipient may alsoinclude a charged lipid and/or detergent in the pharmaceuticalcompositions. Suitable charged lipids include, without limitation,phosphatidylcholines (lecithin), and the like. Detergents will typicallybe a nonionic, anionic, cationic or amphoteric surfactant. Examples ofsuitable surfactants include, for example, Tergitol® and Triton®surfactants (Union Carbide Chemicals and Plastics, Danbury, Conn.),polyoxyethylenesorbitans, for example, TWEEN® surfactants (AtlasChemical Industries, Wilmington, Del.), polyoxyethylene ethers, forexample, Brij, pharmaceutically acceptable fatty acid esters, forexample, lauryl sulfate and salts thereof (SDS), and the like. Suchmaterials may be used as stabilizers and/or anti-oxidants. Additionallythey may be used to reduce local irritation at the site ofadministration.

[0082] In one broad embodiment, the step of administering a drugparticle according to the present invention may include other steps.These other steps may be carried out before, in conjunction with and/orafter the step of administering the drug particle according to theinvention. In one embodiment, these other steps may include applyingtopical medications, for example aluminum chloride; applying aniontophoresis procedure; administering anticholinergics orally orsystemically. In severe hyperhidrosis, other steps may include surgicalmanagement and/or sympathectomy.

[0083] The following examples demonstrate how the various conditions ofhyperhidrosis may be treated according to the present invention.Although particular doses are described, the dose administered can varywidely according to the severity of the condition and other variouspatient variables including size, weight, age, and responsiveness totherapy.

[0084] The examples also show how a neurotoxin or components thereof maybe recombinantly synthesized and reconstituted. The examples relating torecombinant synthesis are substantially similar to the Examples ofInternational Patent Application Publication WO 95/32738, the disclosureof which is incorporated in its entirety herein by reference.

EXAMPLE 1

[0085] Treatment of Gustatory Sweating

[0086] Gustatory sweating (Frey's syndrome, auriculotemporal syndrome)is sweating of the facial skin during meals and commonly is seenfollowing parotid gland surgery and trauma to the preauricular region.Denervated sweat glands become reinnervated by misdirected sprouting ofparasympathetic secretomotor fibers that have lost their “target organ,”the salivary gland. Gustatory sweating is experienced by 13-50% ofpatients after pariodectomy.

[0087] A 40 year old man presents with a classic case of Frey'ssyndrome. The area of hyperhidrosis on the face is visualized by meansof an iodinestarch solution (Minor's iodine-starch test) after sweatingis stimulated by having the patient chew an apple or sour fruit candy.The hyperhidrosis area is then marked with a pen.

[0088] Botulinum toxin type A coated on gold particle carrier is loadedinto a needleless injector. The projection pressure is set so that thedrug particles, i.e., the botulinum toxin A coated gold particles, maybe delivered to the dermis layer of the skin. Also, such an amount ofthe drug particle is loaded so that about 20 U to about 60 U ofbotulinum toxin type A is delivered to 8×10 cm² of the demarcated skinarea. The particular dose of the neurotoxin and area of injection, aswell as the frequency of toxin administrations depend upon a variety offactors to be determined by the treating physician, as previously setforth.

[0089] Seven days after treatment, the gustatory sweating is measuredusing the Minor's iodine test. The hyperhidrotic area shows about a 93%reduction. The reduction in gustatory sweating starts after about 72hours and remains up to about 12 months.

EXAMPLE 2

[0090] Treatment of Axillary Hyperhidrosis

[0091] Axillary hyperhidrosis is a condition which may be socially andemotionally disturbing. It is a condition of excessive sweating, whichmay even cause staining and decaying of clothes. Initially, thetreatment usually consists of topical application of antiperspirantscontaining aluminium salts and/or tanning agents. lontophoresis usingspecial axillary electrodes are also employed in the treatment ofaxillary hyperhidrosis. Oral sedatives, tranquillizers oranticholinergic drugs are sometimes used as an adjunct.

[0092] If the medical treatment proves ineffective or producesunacceptable side-effect, removal of the axillary sweat glands bysurgical excision or liposuction is the other current option. Surgeryand liposuction, although often effective in controlling excessivesweating, are commonly complicated by infection, bleeding, scarring,loss of axillary hair, hypoaesthesia, pain due to nerve injury orentrapment and, occasionally, reinnervation of the residual glands andrecurrence of hyperhidrosis. Denervation of sweat glands bysympathectomy is also effective but carries the risk of pneumothorax,Homer's syndrome and other complications.

[0093] A 35 year old office female dancer presents with a severe case ofaxillary hyperhidrosis. The area of hyperhidrosis under the forearm isvisualized by means of an iodinestarch solution (Minor's iodine-starchtest). The hyperhidrosis area is then marked with a pen.

[0094] Botulinum toxin type A coated on crystal ice particle carrier isloaded into a needleless injector. The projection pressure is set sothat the drug particles, i.e., the botulinum toxin A coated ice crystalparticles, may be delivered to the dermis layer of the skin. Also, suchan amount of the drug particle is loaded so that about 20 U to about 60of botulinum toxin type A is delivered to 8×15 cm² of the demarcatedskin area. The particular dose of the neurotoxin and area of injection,as well as the frequency of toxin administrations depend upon a varietyof factors to be determined by the treating physician, as previously setforth.

[0095] Two weeks after treatment, the axillary sweating response ismeasured using the Minor's iodine test. The hyperhidrotic area showsabout a 95% reduction. The reduction in axillary sweating remains up toabout 27 months, preferably 11 months.

EXAMPLE 3

[0096] Treatment of Palmar Hyperhidrosis

[0097] Botulinum toxin has been injected into the palmar area to treatpalmar hyperhidrosis, and has been found to be very effective. However,one of the main drawback of this treatment is the pain cause by theinjection. The free nerve endings responsible for the pain sensationoccur in the papillary dermis and epidermis whereas the sweat glands areimbedded deep in the dermis and in the upper layer of the subcutaneoustissue. To deliver the botulinum toxin as close to the sweat glands aspossible, subdermal/subcutaneous injections would be optimal, andpresumably less painful than more superficial injections. However, thedeeper the injection the greater the risk of causing weakness of thesmall muscles of the hand and weakening the grip.

[0098] A 22 year old concert pianist presents with a palmarhyperhidrosis. The specific area of hyperhidrosis on the hand isvisualized by means of an iodinestarch solution (Minor's iodine-starchtest). The hyperhidrosis area is then marked with a pen.

[0099] Botulinum toxin type A coated on crystal ice particle carrier isloaded into a needleless injector. The projection pressure is set sothat the drug particles, i.e., the botulinum toxin A coated ice crystalparticles, may be delivered to the dermis layer of the skin. Also, suchamount of the drug particle is loaded so that about 10 U to about 50 Uof botulinum toxin type A is delivered to 10×15 cm² of the demarcatedskin area. An effective therapeutic dose of botulinum toxin is injectedwithout substantial pain. Additionally, no substantial muscle weaknessor fatigue of the hand is observed. The particular dose of theneurotoxin and area of injection, as well as the frequency of toxinadministrations depend upon a variety of factors to be determined by thetreating physician, as previously set forth.

[0100] Two weeks after treatment, the reduced sweating response ismeasured in the area of hyperhidrosis using the Minor's iodine test. Thehyperhidrotic area shows about a 95% reduction. The reduction insweating remains up to about 12 months.

EXAMPLE 4

[0101] Subcloning the BONT/A-L Chain Gene

[0102] This Example describes the methods to clone the polynucleotidesequence encoding the BoNT/A-L chain. The DNA sequence encoding theBoNT/A-L chain is amplified by a PCR protocol that employs syntheticoligonucleotides having the sequences, 5′-AAAGGCCTTTTGTTMTAAACAA-3′ (SEQID#10) and 5′-GGAATTCTTACTTATTGTATCCTTTA-3′ (SEQ ID#11). Use of theseprimers allows the introduction of Stu I and EcoR I restriction sitesinto the 5′ and 3′ ends of the BoNT/A-L chain gene fragment,respectively. These restriction sites are subsequently used tofacilitate unidirectional subcloning of the amplification products.Additionally, these primers introduce a stop codon at the C-terminus ofthe L chain coding sequence. Chromosomal DNA from C. botulinum (strain63 A) serves as a template in the amplification reaction.

[0103] The PCR amplification is performed in a 100 μl volume containing10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl₂, 0.2 mM of eachdeoxynucleotide triphosphate (dNTP), 50 pmol of each primer, 200 ng ofgenomic DNA and 2.5 units of Taq-polymerase (Promega). The reactionmixture is subjected to 35 cycles of denaturation (1 minute at 94° C.),annealing (2 minutes at 37° C.) and polymerization (2 minutes at 72°C.). Finally, the reaction is extended for an additional 5 minutes at72° C.

[0104] The PCR amplification product is digested with Stu I and EcoR I,purified by agarose gel electrophoresis, and ligated into Sma I and EcoRI digested pBluescript II SK* to yield the plasmid, pSAL. Bacterialtransformants harboring this plasmid are isolated by standardprocedures. The identity of the cloned L chain polynucleotide isconfirmed by double stranded plasmid sequencing using SEQUENASE (UnitedStates Biochemicals) according to the manufacturer's instructions.Synthetic oligonucleotide sequencing primers are prepared as necessaryto achieve overlapping sequencing runs. The cloned sequence is found tobe identical to the sequence disclosed by Binz, et al., in J. Biol.Chem. 265:9153 (1990), and Thompson et al., in Eur. J. Biochem. 189:73(1990).

[0105] Site-directed mutants designed to compromise the enzymaticactivity of the BoNT/A-L chain can also be created.

EXAMPLE 5

[0106] Expression of the Botulinum Toxin Type A-L (BoNt/VA-L) ChainFusion Proteins

[0107] This Example describes the methods to verify expression of thewild-type L chains, which may serve as a therapeutic component, inbacteria harboring the pCA-L plasmids. Well isolated bacterial coloniesharboring either pCAL are used to inoculate L-broth containing 100 μg/mlampicillin and 2% (w/v) glucose, and grown overnight with shaking at 30°C. The overnight cultures are diluted 1:10 into fresh L-broth containing100 μg/ml of ampicillin and incubated for 2 hours. Fusion proteinexpression is induced by addition of IPTG to a final concentration of0.1 mM. After an additional 4 hour incubation at 30° C., bacteria arecollected by centrifugation at 6,000×g for 10 minutes.

[0108] A small-scale SDS-PAGE analysis confirmed the presence of a 90kDa protein band in samples derived from IPTG-induced bacteria. ThisM_(r) is consistent with the predicted size of a fusion protein havingMBP (˜40 kDa) and BoNT/A-L chain (˜50 kDa) components. Furthermore, whencompared with samples isolated from control cultures, the IPTG-inducedclones contained substantially larger amounts of the fusion protein.

[0109] The presence of the desired fusion proteins in IPTG-inducedbacterial extracts is also confirmed by Western blotting using thepolyclonal anti-L chain probe described by Cenci di Bello et al., inEur. J. Biochem. 219:161 (1993). Reactive bands on PVDF membranes(Pharmacia; Milton Keynes, UK) are visualized using an anti-rabbitimmunoglobulin conjugated to horseradish peroxidase (Bio-Rad; HemelHempstead, UK) and the ECL detection system (Amersham, UK). Westernblotting results confirmed the presence of the dominant fusion proteintogether with several faint bands corresponding to proteins of lowerM_(r) than the fully sized fusion protein. This observation suggestedthat limited degradation of the fusion protein occurred in the bacteriaor during the isolation procedure. Neither the use of 1 mM nor 10 mMbenzamidine (Sigma; Poole, UK) during the isolation procedure eliminatedthis proteolytic breakdown.

[0110] The yield of intact fusion protein isolated by the aboveprocedure remained fully adequate for ell procedures described herein.Based on estimates from stained SDS-PAGE gels, the bacterial clonesinduced with IPTG yielded 5-10 mg of total MBP-wild-type or mutant Lchain fusion protein per liter of culture. Thus, the method of producingBoNT/A-L chain fusion proteins disclosed herein is highly efficient,despite any limited proteolysis that did occur.

[0111] The MBP-L chain fusion proteins encoded by the PCAL andPCAL-TyrU7 expression plasmids are purified from bacteria by amyloseaffinity chromatography. Recombinant wild-type or mutant L chains arethen separated from the sugar binding domains of the fusion proteins bysite-specific cleavage with Factor X₂. This cleavage procedure yieldedfree MBP, free L chains and a small amount of uncleaved fusion protein.While the resulting L chains present in such mixtures have been shown topossess the desired activities, we have also employed an additionalpurification step. Accordingly, the mixture of cleavage products isapplied to a second amylose affinity column that bound both the MBP anduncleaved fusion protein. Free L chains are not retained on the affinitycolumn, and are isolated for use in experiments described below.

EXAMPLE 6

[0112] Purification of Fusion Proteins and Isolation of RecombinantBoNT/A-L Chains

[0113] This Example describes a method to produce and purify wild-typerecombinant BoNT/A light chains from bacterial clones. Pellets from 1liter cultures of bacteria expressing the wild-type BoNT/A-L chainproteins are resuspended in column buffer [10 mM Tris-HCl (pH 8.0), 200mM NaCl, 1 mM EGTA and 1 mM DTT] containing 1 mM phenyl-methanesulfonylfluoride (PMSF) and 10 mM benzamidine, and lysed by sonication. Thelysates are cleared by centrifugation at 15,000×g for 15 minutes at 4°C. Supernatants are applied to an amylose affinity column [2×10 cm, 30ml resin] (New England BioLabs; Hitchin, UK). Unbound proteins arewashed from the resin with column buffer until the eluate is free ofprotein as judged by a stable absorbance reading at 280 nm. The boundMBP-L chain fusion protein is subsequently eluted with column buffercontaining 10 mM maltose. Fractions containing the fusion protein arepooled and dialyzed against 20 mM Tris-HCl (pH 8.0) supplemented with150 mM NaCl, 2 mM, CaCI₂ and 1 mM DTT for 72 hours at 4° C.

[0114] Fusion proteins are cleaved with Factor X₂ (Promega; Southampton,UK) at an enzyme:substrate ratio of 1:100 while dialyzing against abuffer of 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM,CaCl₂ and 1 mM DTT. Dialysis is carried out for 24 hours at 4° C. Themixture of MBP and either wild-type or mutant L chain that resulted fromthe cleavage step is loaded onto a 10 ml amylose column equilibratedwith column buffer. Aliquots of the flow through fractions are preparedfor SDS-PAGE analysis to identify samples containing the L chains.Remaining portions of the flow through fractions are stored at −20° C.Total E. coli extract or the purified proteins are solubilized in SDSsample buffer and subjected to PAGE according to standard procedures.Results of this procedure indicated the recombinant toxin fragmentaccounted for roughly 90% of the protein content of the sample.

[0115] The foregoing results indicates that the approach to creatingMBP-L chain fusion proteins described herein could be used toefficiently produce wild-type and mutant recombinant BoNT/A-L chains.Further, the results demonstrate that recombinant L chains could beseparated from the maltose binding domains of the fusion proteins andpurified thereafter.

[0116] A sensitive antibody-based assay is developed to compare theenzymatic activities of recombinant L chain products and their nativecounterparts. The assay employed an antibody having specificity for theintact C-terminal region of SNAP-25 that corresponded to the BoNT/Acleavage site. Western Blotting of the reaction products of BoNT/Acleavage of SNAP-25 indicated an inability of the antibody to bindSNAP-25 sub-fragments. Thus, the antibody reagent employed in thefollowing Example detected only intact SNAP-25. The loss of antibodybinding served as an indicator of SNAP-25 proteolysis mediated by addedBoNT/A light chain or recombinant derivatives thereof.

EXAMPLE 7

[0117] Evaluation of the Proteolytic Activities of Recombinant L ChainsAgainst a SNAP-25 Substrate

[0118] This Example describes a method to demonstrate that both nativeand recombinant BoNT/A-L chains can proteolyze a SNAP-25 substrate. Aquantitative assay is employed to compare the abilities of the wild-typeand their recombinant analogs to cleave a SNAP-25 substrate. Thesubstrate utilized for this assay is obtained by preparing aglutathione-S-transferase (GST)-SNAP-25 fusion protein, containing acleavage site for thrombin, expressed using the pGEX-2T vector andpurified by affinity chromatography on glutathione agarose. The SNAP-25is then cleaved from the fusion protein using thrombin in 50 mM Tris-HCl(pH 7.5) containing 150 mM NaCl and 2.5 mM CaCl₂ (Smith et al., Gene67:31 (1988)) at an enzyme:substrate ratio of 1:100. Uncleaved fusionprotein and the cleaved glutathione-binding domain bound to the gel. Therecombinant SNAP-25 protein is eluted with the latter buffer anddialyzed against 100 mM HEPES (pH 7.5) for 24 hours at 4° C. The totalprotein concentration is determined by routine methods.

[0119] Rabbit polyclonal antibodies specific for the C-terminal regionof SNAP-25 are raised against a synthetic peptide having the amino acidsequence, CANQRATKMLGSG (SEQ ID#12). This peptide corresponded toresidues 195 to 206 of the synaptic plasma membrane protein and anN-terminal cysteine residue not found in native SNAP-25. The syntheticpeptide is conjugated to bovine serum albumin (BSA) (Sigma; Poole, UK)using maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as across-linking agent (Sigma; Poole, UK) to improve antigenicity (Liu etal., Biochemistry 18:690 (1979)1. Affinity purification of theanti-peptide antibodies is carried out using a column having theantigenic peptide conjugated via its N-terminal cysteine residue to anaminoalkyl agarose resin (Bio-Rad; Hemel Hempstead, UK), activated withiodoacetic acid using the cross-linker ethyl 3-(3-dimethytpropyl)carbodiimide. After successive washes of the column with a buffercontaining 25 mM Tris-HCl (pH 7.4) and 150 mM NaCl, the peptide-specificantibodies are eluted using a solution of 100 mM glycine (pH 2.5) and200 mM NaCl, and collected in tubes containing 0.2 ml of 1 M Tris-HCl(pH 8.0) neutralizing buffer.

[0120] All recombinant preparations containing wild-type L chain aredialyzed overnight at 4° C. into 100 mM HEPES (pH 7.5) containing 0.02%Lubrol and 10 μM zinc acetate before assessing their enzymaticactivities. BoNT/A, previously reduced with 20 mM DTT for 30 minutes at37° C., as well as these dialyzed samples, are then diluted to differentconcentrations in the latter HEPES buffer supplemented with 1 mM DTT.

[0121] Reaction mixtures include 5 μl recombinant SNAP-25 substrate (8.5μM final concentration) and either 20 μl reduced BoNT/A or recombinantwild-type L chain. All samples are incubated at 37° C. for 1 hour beforequenching the reactions with 25 μl aqueous 2% trifluoroacetic acid (TFA)and 5 mM EDTA (Foran et al., Biochemistry 33:15365(1994)). Aliquots ofeach sample are prepared for SDS-PAGE and Western blotting with thepolyclonal SNAP-25 antibody by adding SDS-PAGE sample buffer andboiling. Anti-SNAP-25 antibody reactivity is monitored using an ECLdetection system and quantified by densitometric scanning.

[0122] Western blotting results indicate clear differences between theproteolytic activities of the purified mutant L chain and either nativeor recombinant wild-type BoNT/A-L chain. Specifically, recombinantwild-type L chain cleaves the SNAP-25 substrate, though somewhat lessefficiently than the reduced BoNT/A native L chain that serves as thepositive control in the procedure. Thus, an enzymatically active form ofthe BoNT/A-L chain is produced by recombinant means and subsequentlyisolated. Moreover, substitution of a single amino acid in the L chainprotein abrogated the ability of the recombinant protein to degrade thesynaptic terminal protein. As a preliminary test of the biologicalactivity of the wild-type recombinant BoNT/A-L chain, the ability of theMBP-L chain fusion protein to diminish Ca²⁺-evoked catecholamine releasefrom digitonin-permeabilized bovine adrenochromaffin cells is examined.Consistently, wild-type recombinant L chain fusion protein, eitherintact or cleaved with Factor X₂ to produce a mixture containing freeMBP and recombinant L chain, induced a dose-dependent inhibition ofCa²⁺-stimulated release equivalent to the inhibition caused by nativeBoNT/A.

EXAMPLE 8

[0123] Reconstitution of Native L Chain, Recombinant Wild-type L Chainwith Purified H Chain

[0124] Native H and L chains are dissociated from BoNT/A (ListBiologicals Inc.; Campbell, USA) with 2 M urea, reduced with 100 mM DTTand then purified according to established chromatographic procedures(Kozaki et al., Japan J. Med. Sci. Biol. 34:61 (1981); Maisey et al.,Eur. J. Biochem. 177:683 (1988)). Purified H chain is combined with anequimolar amount of either native L chain or recombinant wild-type Lchain. Reconstitution is carried out by dialyzing the samples against abuffer consisting of 25 mM Tris (pH 8.0), 50 μM zinc acetate and 150 mMNaCl over 4 days at 4° C. Following dialysis, the association of therecombinant L chain and native H chain to form disulfide-linked 150 kDadichains is monitored by SDS-PAGE and quantified by densitometricscanning. The proportion of dichain molecules formed with therecombinant L chains is lower than that obtained when native L chain isemployed. Indeed, only about 30% of the recombinant wild-type or mutantL chain is reconstituted while >90% of the native L chain reassociatedwith the H chain. In spite of this lower efficiency of reconstitution,sufficient material incorporating the recombinant L chains is easilyproduced for use in subsequent functional studies.

[0125] While this invention has been described with respect to variousspecific examples and embodiments, it is to be understood that theinvention is not limited thereto and that it can be variously practicedwith the scope of the following claims. Other embodiments, versions, andmodifications within the scope of the present invention are possible.

1 12 1 3891 DNA botulinum toxin 1 atgcaatttg ttaataaaca atttaattataaagatcctg taaatggtgt tgatattgct 60 tatataaaaa ttccaaatgt aggacaaatgcaaccagtaa aagcttttaa aattcataat 120 aaaatatggg ttattccaga aagagatacatttacaaatc ctgaagaagg agatttaaat 180 ccaccaccag aagcaaaaca agttccagtttcatattatg attcaacata tttaagtaca 240 gataatgaaa aagataatta tttaaagggagttacaaaat tatttgagag aatttattca 300 actgatcttg gaagaatgtt gttaacatcaatagtaaggg gaataccatt ttggggtgga 360 agtacaatag atacagaatt aaaagttattgatactaatt gtattaatgt gatacaacca 420 gatggtagtt atagatcaga agaacttaatctagtaataa taggaccctc agctgatatt 480 atacagtttg aatgtaaaag ctttggacatgaagttttga atcttacgcg aaatggttat 540 ggctctactc aatacattag atttagcccagattttacat ttggttttga ggagtcactt 600 gaagttgata caaatcctct tttaggtgcaggcaaatttg ctacagatcc agcagtaaca 660 ttagcacatg aacttataca tgctggacatagattatatg gaatagcaat taatccaaat 720 agggttttta aagtaaatac taatgcctattatgaaatga gtgggttaga agtaagcttt 780 gaggaactta gaacatttgg gggacatgatgcaaagttta tagatagttt acaggaaaac 840 gaatttcgtc tatattatta taataagtttaaagatatag caagtacact taataaagct 900 aaatcaatag taggtactac tgcttcattacagtatatga aaaatgtttt taaagagaaa 960 tatctcctat ctgaagatac atctggaaaattttcggtag ataaattaaa atttgataag 1020 ttatagaaaa tgttaacaga gatttagacagaggataatt ttgttaagtt ttttaaagta 1080 cttaacagaa aaacatattt gaattttgataaagccgtat ttaagataaa tatagtacct 1140 aaggtaaatt acacaatata tgatggatttaatttaagaa atacaaattt agcagcaaac 1200 tttaatggtc aaaatacaga aattaataatatgaatttta ctaaactaaa aaattttact 1260 ggattgtttg aattttataa gttgctatgtgtaagaggga taataacttc taaaactaaa 1320 tcattagata aaggatacaa taaggcattaaatgatttat gtatcaaagt taataattgg 1380 gacttgtttt ttagtccttc agaagataattttactaatg atctaaataa aggagaagaa 1440 attacatctg atactaatat agaagcagcagaagaaaata ttagtttaga tttaatacaa 1500 caatattatt taacctttaa ttttgataatgaacctgaaa atatttcaat agaaaatctt 1560 tcaagtgaca ttataggcca attagaacttatgcctaata tagaaagatt tcctaatgga 1620 aaaaagtatg agttagataa atatactatgttccattatc ttcgtgctca agaatttgaa 1680 catggtaaat ctaggattgc tttaacaaattctgttaacg aagcattatt aaatcctagt 1740 cgtgtttata catttttttc ttcagactatgtaaagaaag ttaataaagc tacggaggca 1800 gctatgtttt taggctgggt agaacaattagtatatgatt ttaccgatga aactagcgaa 1860 gtaagtacta cggataaaat tgcggatataactataatta ttccatatat aggacctgct 1920 ttaaatatag gtaatatgtt atataaagatgattttgtag gtgctttaat attttcagga 1980 gctgttattc tgttagaatt tataccagagattgcaatac ctgtattagg tacttttgca 2040 cttgtatcat atattgcgaa taaggttctaaccgttcaaa caatagataa tgctttaagt 2100 aaaagaaatg aaaaatggga tgaggtctataaatatatag taacaaattg gttagcaaag 2160 gttaatacac agattgatct aataagaaaaaaaatgaaag aagctttaga aaatcaagca 2220 gaagcaacaa aggctataat aaactatcagtataatcaat atactgagga agagaaaaat 2280 aatattaatt ttaatattga tgatttaagttcgaaactta atgagtctat aaataaagct 2340 atgattaata taaataaatt tttgaatcaatgctctgttt catatttaat gaattctatg 2400 atcccttatg gtgttaaacg gttagaagattttgatgcta gtcttaaaga tgcattatta 2460 aagtatatat atgataatag aggaactttaattggtcaag tagatagatt aaaagataaa 2520 gttaataata cacttagtac agatataccttttcagcttt ccaaataggt agataatcaa 2580 agattattat ctacatttac tgaatatattaagaatatta ttaatacttc tatattgaat 2640 ttaagatatg aaagtaatca tttaatagacttatctaggt atgcatcaaa aataaatatt 2700 ggtagtaaag taaattttga tccaatagataaaaatcaaa ttcaattatt taatttagaa 2760 agtagtaaaa ttgaggtaat tttaaaaaatgctattgtat ataatagtat gtatgaaaat 2820 tttagtacta gcttttggat aagaattcctaagtatttta acagtataag tctaaataat 2880 gaatatacaa taataaattg tatggaaaataattcaggat ggaaagtatc acttaattat 2940 ggtgaaataa tctggacttt acaggatactcaggaaataa aacaaagagt agtttttaaa 3000 tagagtcaaa tgattaatat atcagattatataaacagat ggatttttgt aactatcact 3060 aataatagat taaataactc taaaatttatataaatggaa gattaataga tgaaaaacca 3120 atttcaaatt taggtaatat tcatgctagtaataatataa tgtttaaatt agatggttgt 3180 agagatacac atagatatat ttggataaaatattttaatc tttttgataa ggaattaaat 3240 gaaaaagaaa tcaaagattt atatgataatcaatcaaatt caggtatttt aaaagacttt 3300 tggggtgatt atttacaata tgataaaccatagtatatgt taaatttata tgatccaaat 3360 aaatatgtcg atgtaaataa tgtaggtattagaggttata tgtatcttaa agggcctaga 3420 ggtagcgtaa tgactacaaa catttatttaaattcaagtt tgtatagggg gacaaaattt 3480 attataaaaa aatatgcttc tggaaataaagataatattg ttagaaataa tgatcgtgta 3540 tatattaatg tagtagttaa aaataaagaatataggttag ctactaatgc atcacaggca 3600 ggcgtagaaa aaatactaag tgcattagaaatacctgatg taggaaatct aagtcaagta 3660 gtagtaatga agtcaaaaaa tgatcaaggaataacaaata aatgcaaaat gaatttacaa 3720 gataataatg ggaatgatat aggctttataggatttcatc agtttaataa tatagctaaa 3780 ctagtagcaa gtaattggta taatagacaaatagaaagat ctagtaggac tttgggttgc 3840 tcatgggaat ttattcctgt agatgatggatggggagaaa ggccactgta a 3891 2 3876 DNA botulinum toxin 2 atgccagttacaataaataa ttttaattat aatgatccta ttgataatga caatattatt 60 atgatggaacctccatttgc aaggggtacg gggagatatt ataaagcttt taaaatcaca 120 gatcgtatttggataatacc cgaaagatat acttttggat ataaacctga ggattttaat 180 aaaagttccggtatttttaa tagagatgtt tgtgaatatt atgatccaga ttacttaaat 240 accaatgataaaaagaatat atttttccaa acattgatca agttatttaa tagaatcaaa 300 tcaaaaccattgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga 360 gatagacgtgttccactcga agagtttaac acaaacattg ctagtgtaac tgttaataaa 420 ttaattagtaatccaggaga agtggagcga aaaaaaggta ttttcgcaaa tttaataata 480 tttggacctgggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat 540 tttgcatcaagggaaggctt tgggggtata atgcaaatga aattttgtcc agaatatgta 600 agcgtatttaataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat 660 ttttcagatccagccttgat attaatgcat gaacttatac atgttttgca tggattatat 720 ggcattaaagtagatgattt accaattgta ccaaatgaaa aaaaattttt tatgcaatct 780 acagatactatacaggcaga agaactatat acatttggag gacaagatcc cagcatcata 840 tctccttctacagataaaag tatctatgat aaagttttgc aaaattttag ggggatagtt 900 gatagacttaacaaggtttt agtttgcata tcagatccta acattaacat taatatatat 960 aaaaataaatttaaagataa atataaattc gttgaagatt ctgaaggaaa atatagtata 1020 gatgtagaaagtttcaataa attatataaa agcttaatgt taggttttac agaaattaat 1080 atagcagaaaattataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca 1140 gtaaaaataaaaaatttatt agataatgaa atctatacta tagaggaagg gtttaatata 1200 tctgataaaaatatgggaaa agaatatagg ggtcagaata aagctataaa taaacaagct 1260 tatgaagaaatcagcaagga gcatttggct gtatataaga tacaaatgtg taaaagtgtt 1320 aaagttccaggaatatgtat tgatgtcgat aatgaaaatt tgttctttat agctgataaa 1380 aatagtttttcagatgattt atctaaaaat gaaagagtag aatataatac acagaataat 1440 tatataggaaatgactttcc tataaatgaa ttaattttag atactgattt aataagtaaa 1500 atagaattaccaagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta 1560 tatgaaaaacaacccgctat aaaaaaagtt tttacagatg aaaataccat ctttcaatat 1620 ttatactctcagacatttcc tctaaatata agagatataa gtttaacatc ttcatttgat 1680 gatgcattattagtttctag caaagtttat tcattttttt ctatggatta tattaaaact 1740 gctaataaagtagtagaagc aggattattt gcaggttggg tgaaacagat agtagatgat 1800 tttgtaatcgaagctaataa aagcagtact atggataaaa ttgcagatat atctctaatt 1860 gttccttatataggattagc tttaaatgta ggagatgaaa cagctaaagg aaattttgaa 1920 agtgcttttgagattgcagg atccagtatt ttactagaat ttataccaga acttttaata 1980 cctgtagttggagtcttttt attagaatca tatattgaca ataaaaataa aattattaaa 2040 acaatagataatgctttaac taaaagagtg gaaaaatgga ttgatatgta gggattaata 2100 gtagcgcaatggctctcaac agttaatact caattttata caataaaaga gggaatgtat 2160 aaggctttaaattatcaagc acaagcattg gaagaaataa taaaatacaa atataatata 2220 tattctgaagaggaaaagtc aaatattaac atcaatttta atgatataaa ttctaaactt 2280 aatgatggtattaaccaagc tatggataat ataaatgatt ttataaatga atgttctgta 2340 tcatatttaatgaaaaaaat gattccatta gctgtaaaaa aattactaga ctttgataat 2400 actctcaaaaaaaatttatt aaattatata gatgaaaata aattatattt aattggaagt 2460 gtagaagatgaaaaatcaaa agtagataaa tacttgaaaa ccattatacc atttgatctt 2520 tcaacgtattctaatattga aatactaata aaaatattta ataaatataa tagcgaaatt 2580 ttaaataatattatcttaaa tttaagatat agagataata atttaataga tttatcagga 2640 tatggagcaaaggtagaggt atatgatggg gtcaagctta atgataaaaa tcaatttaaa 2700 ttaactagttcagcagatag taagattaga gtcactcaaa atcagaatat tatatttaat 2760 agtatgttccttgattttag cgttagcttt tggataagga tacctaaata taggaatgat 2820 gatatacaaaattatattca taatgaatat acgataatta attgtatgaa aaataattca 2880 ggctggaaaatatctattag gggtaatagg ataatatgga ccttaattga tataaatgga 2940 aaaaccaaatcagtattttt tgaatataac ataagagaag atatatcaga gtatataaat 3000 agatggttttttgtaactat tactaataat ttggataatg ctaaaattta tattaatggc 3060 acgttagaatcaaatatgga tattaaagat ataggagaag ttattgttaa tggtgaaata 3120 acatttaaattagatggtga tgtagataga acacaattta tttggatgaa atattttagt 3180 atttttaatacgcaattaaa tcaatcaaat attaaagaga tatataaaat tcaatcatat 3240 agcgaatagttaaaagattt ttggggaaat cctttaatgt ataataaaga atattatatg 3300 tttaatgcggggaataaaaa ttcatatatt aaactagtga aagattcatc tgtaggtgaa 3360 atattaatacgtagcaaata taatcagaat tccaattata taaattatag aaatttatat 3420 attggagaaaaatttattat aagaagagag tcaaattctc aatctataaa tgatgatata 3480 gttagaaaagaagattatat acatctagat ttggtacttc accatgaaga gtggagagta 3540 tatgcctataaatattttaa ggaacaggaa gaaaaattgt ttttatctat tataagtgat 3600 tctaatgaattttataagac tatagaaata aaagaatatg atgaacagcc atcatatagt 3660 tgtcagttgctttttaaaaa agatgaagaa agtactgatg atataggatt gattggtatt 3720 catcgtttctaggaatctgg agttttacgt aaaaagtata aagattattt ttgtataagt 3780 aaatggtagttaaaagaggt aaaaaggaaa ccatataagt caaatttggg atgtaattgg 3840 cagtttattcctaaagatga agggtggact gaataa 3876 3 3876 DNA botulinum toxin 3atgccagtta caataaataa ttttaattat aatgatccta ttgataataa taatattatt 60atgatggagc ctccatttgc gagaggtacg gggagatatt ataaagcttt taaaatcaca 120gatcgtattt ggataatacc ggaaagatat acttttggat ataaacctga ggattttaat 180aaaagttccg gtatttttaa tagagatgtt tgtgaatatt atgatccaga ttagttaaat 240actaatgata aaaagaatat atttttacaa acaatgatca agttatttaa tagaatcaaa 300tcaaaaccat tgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga 360gatagacgtg ttccactcga agagtttaac acaaacattg ctagtgtaac tgttaataaa 420ttaatcagta atccaggaga agtggagcga aaaaaaggta ttttcgcaaa tttaataata 480tttggacctg ggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat 540tttgcatcaa gggaaggctt cgggggtata atgcaaatga agttttgccc agaatatgta 600agcgtattta ataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat 660ttttcagatc cagccttgat attaatgcat gaacttatac atgttttaca tggattatat 720ggcattaaag tagatgattt accaattgta ccaaatgaaa aaaaattttt tatgcaatct 780acagatgcta tacaggcaga agaactatat acatttggag gacaagatcc cagcatcata 840actccttcta cggataaaag tatctatgat aaagttttgc aaaattttag agggatagtt 900gatagactta acaaggtttt agtttgcata tcagatccta acattaatat taatatatat 960aaaaataaat ttaaagataa atataaattc gttgaagatt ctgagggaaa atatagtata 1020gatgtagaaa gttttgataa attatataaa agcttaatgt ttggttttac agaaactaat 1080atagcagaaa attataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca 1140gtaaaaataa aaaatttatt agataatgaa atctatacta tagaggaagg gtttaatata 1200tctgataaag atatggaaaa agaatataga ggtcagaata aagctataaa taaacaagct 1260tatgaagaaa ttagcaagga gcatttggct gtatataaga tacaaatgtg taaaagtgtt 1320aaagctccag gaatatgtat tgatgttgat aatgaagatt tgttctttat agctgataaa 1380aatagttttt cagatgattt atctaaaaac gaaagaatag aatataatac acagagtaat 1440tatatagaaa atgacttccc tataaatgaa ttaattttag atactgattt aataagtaaa 1500atagaattac caagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta 1560tatgaaaaac aacccgctat aaaaaaaatt tttacagatg aaaataccat ctttcaatat 1620ttatagtctc agacatttct cttagatata agagatataa gtttaacatc ttcatttgat 1680gatgcattat tattttctaa caaagtttat tcattttttt ctatggatta tattaaaact 1740gctaataaag tggtagaagc aggattattt gcaggttggg tgaaacagat agtaaatgat 1800tttgtaatcg aagctaataa aagcaatact atggataaaa ttgcagatat atctctaatt 1860gttccttata taggattagc tttaaatgta ggaaatgaaa cagctaaagg aaattttgaa 1920aatgcttttg agattgcagg agccagtatt ctactagaat ttataccaga acttttaata 1980cctgtagttg gagccttttt attagaatca tatattgaca ataaaaataa aattattaaa 2040acaatagata atgctttaac taaaagaaat gaaaaatgga gtgatatgta gggattaata 2100gtagcgcaat ggctctcaac agttaatact caattttata caataaaaga gggaatgtat 2160aaggctttaa attatcaagc acaagcattg gaagaaataa taaaatacag atataatata 2220tattctgaaa aagaaaagtc aaatattaac atcgatttta atgatataaa ttctaaactt 2280aatgagggta ttaaccaagc tatagataat ataaataatt ttataaatgg atgttctgta 2340tcatatttaa tgaaaaaaat gattccatta gctgtagaaa aattactaga ctttgataat 2400actctcaaaa aaaatttgtt aaattatata gatgaaaata aattatattt gattggaagt 2460gcagaatatg aaaaatcaaa agtaaataaa tagttgaaaa ccattatgcc gtttgatctt 2520tcaatatata ccaatgatac aatactaata gaaatgttta ataaatataa tagcgaaatt 2580ttaaataata ttatcttaaa tttaagatat aaggataata atttaataga tttatcagga 2640tatggggcaa aggtagaggt atatgatgga gtcgagctta atgataaaaa tcaatttaaa 2700ttaactagtt cagcaaatag taagattaga gtgactcaaa atcagaatat catatttaat 2760agtgtgttcc ttgattttag cgttagcttt tggataagaa tacctaaata taagaatgat 2820ggtatacaaa attatattca taatgaatat acaataatta attgtatgaa aaataattcg 2880ggctggaaaa tatctattag gggtaatagg ataatatgga ctttaattga tataaatgga 2940aaaaccaaat cggtattttt tgaatataac ataagagaag atatatcaga gtatataaat 3000agatggtttt ttgtaactat tactaataat ttgaataacg ctaaaattta tattaatggt 3060aagctagaat caaatacaga tattaaagat ataagagaag ttattgctaa tggtgaaata 3120atatttaaat tagatggtga tatagataga acacaattta tttggatgaa atatttcagt 3180atttttaata cggaattaag tcaatcaaat attgaagaaa gatataaaat tcaatcatat 3240agcgaatatt taaaagattt ttggggaaat cctttaatgt agaataaaga atattatatg 3300tttaatgcgg ggaataaaaa ttcatatatt aaactaaaga aagattcacc tgtaggtgaa 3360attttaacac gtagcaaata taatcaaaat tctaaatata taaattatag agatttatat 3420attggagaaa aatttattat aagaagaaag tcaaattctc aatctataaa tgatgatata 3480gttagaaaag aagattatat atatctagat ttttttaatt taaatcaaga gtggagagta 3540tatacctata aatattttaa gaaagaggaa gaaaaattgt ttttagctcc tataagtgat 3600tctgatgagt tttagaatac tatacaaata aaagaatatg atgaacagcc aacatatast 3660tgtcagttgc tttttaaaaa agatgaagaa agtactgatg agataggatt gattggtatt 3720catcgtttct aggaatctgg aattgtattt gaagagtata aagattattt ttgtataagt 3780aaatggtagt taaaagaggt aaaaaggaaa ccatataatt taaaattggg atgtaattgg 3840cagtttattc ctaaagatga agggtggact gaataa 3876 4 3876 DNA botulinum toxin4 atgccaataa caattaacaa ctttaattat tcagatcctg ttgataataa aaatatttta 60tatttagata ctcatttaaa tacactagct aatgagcctg aaaaagcctt tcgcattaca 120ggaaatatat gggtaatacc tgatagattt tcaagaaatt ctaatccaaa tttaaataaa 180cctcctcgag ttacaagccc taaaagtggt tattatgatc ctaattattt gagtactgat 240tctgacaaag atacattttt aaaagaaatt ataaagttat ttaaaagaat taattctaga 300gaaataggag aagaattaat atatagactt tcgacagata taccctttcc tgggaataac 360aatactccaa ttaatacttt tgattttgat gtagatttta acagtgttga tgttaaaact 420agacaaggta acaactgggt taaaactggt agcataaatc ctagtgttat aataactgga 480cctagagaaa acattataga tccagaaact tctacgttta aattaactaa caatactttt 540gcggcacaag aaggatttgg tgctttatca ataatttcaa tatcacctag atttatgcta 600acatatagta atgcaactaa tgatgtagga gagggtagat tttctaagtc tgaattttgc 660atggatccaa tactaatttt aatgcatgaa cttaatcatg caatgcataa tttatatgga 720atagctatac caaatgatca aacaatttca tctgtaacta gtaatatttt ttattctcaa 780tataatgtga aattagagta tgcagaaata tatgcatttg gaggtccaac tatagacctt 840attcctaaaa gtgcaaggaa atattttgag gaaaaggcat tggattatta tagatctata 900gctaaaagac ttaatagtat aactactgca aatccttcaa gctttaataa atatataggg 960gaatataaac agaaacttat tagaaagtat agattcgtag tagaatcttc aggtgaagtt 1020acagtaaatc gtaataagtt tgttgagtta tataatgaac ttacacaaat atttacagaa 1080tttaactagg ctaaaatata taatgtacaa aataggaaaa tatatctttc aaatgtatat 1140actccggtta cggcgaatat attagacgat aatgtttatg atatacaaaa tggatttaat 1200atacctaaaa gtaatttaaa tgtactattt atgggtcaaa atttatctcg aaatccagca 1260ttaagaaaag tcaatcctga aaatatgctt tatttattta caaaattttg tcataaagca 1320atagatggta gatcattata taataaaaca ttagattgta gagagctttt agttaaaaat 1380actgacttac cctttatagg tgatattagt gatgttaaaa ctgatatatt tttaagaaaa 1440gatattaatg aagaaactga agttatatac tatccggaca atgtttcagt agatcaagtt 1500attctcagta agaatacctc agaacatgga caactagatt tattataccc tagtattgac 1560agtgagagtg aaatattacc aggggagaat caagtctttt atgataatag aactcaaaat 1620gttgattatt tgaattctta ttattaccta gaatctcaaa aactaagtga taatgttgaa 1680gattttactt ttacgagatc aattgaggag gctttggata atagtgcaaa agtatatact 1740tactttccta cactagctaa taaagtaaat gcgggtgttc aaggtggttt atttttaatg 1800tgggcaaatg atgtagttga agattttact acaaatattc taagaaaaga tacattagat 1860aaaatatcag atgtatcagc tattattccc tatataggac ccgcattaaa tataagtaat 1920tctgtaagaa gaggaaattt tactgaagca tttgcagtta ctggtgtaac tattttatta 1980gaagcatttc ctgaatttac aatacctgca cttggtgcat ttgtgattta tagtaaggtt 2040caagaaagaa acgagattat taaaactata gataattgtt tagaacaaag gattaagaga 2100tggaaagatt catatgaatg gatgatggga acgtggttat ccaggattat tactcaattt 2160aataatataa gttatcaaat gtatgattct ttaaattatc aggcaggtgc aatcaaagct 2220aaaatagatt tagaatataa aaaatattca ggaagtgata aagaaaatat aaaaagtcaa 2280gttgaaaatt taaaaaatag tttagatgta aaaatttcgg aagcaatgaa taatataaat 2340aaatttatac gagaatgttc cgtaacatat ttatttaaaa atatgttacc taaagtaatt 2400gatgaattaa atgagtttga tcgaaatact aaagcaaaat taattaatct tatagatagt 2460cataatatta ttctagttgg tgaagtagat aaattaaaag caaaagtaaa taatagcttt 2520caaaatacaa taccctttaa tattttttca tatactaata attctttatt aaaagatata 2580attaatgaat atttcaataa tattaatgat tcaaaaattt tgagcctaca aaacagaaaa 2640aatactttag tggatacatc aggatataat gcagaagtga gtgaagaagg cgatgttcag 2700cttaatccaa tatttccatt tgactttaaa ttaggtagtt caggggagga tagaggtaaa 2760gttatagtaa cccagaatga aaatattgta tataattcta tgtatgaaag ttttagcatt 2820agtttttgga ttagaataaa taaatgggta agtaatttac ctggatatac tataattgat 2880agtgttaaaa ataactcagg ttggagtata ggtattatta gtaatttttt agtatttact 2940ttaaaacaaa atgaagatag tgaacaaagt ataaatttta gttatgatat atcaaataat 3000gctcctggat agaataaatg gttttttgta actgttacta acaatatgat gggaaatatg 3060aagatttata taaatggaaa attaatagat actataaaag ttaaagaact aactggaatt 3120aattttagca aaactataac atttgaaata aataaaattc cagataccgg tttgattact 3180tcagattctg ataacatcaa tatgtggata agagattttt atatatttgc taaagaatta 3240gatggtaaag atattaatat attatttaat agcttgcaat atactaatgt tgtaaaagat 3300tattggggaa atgatttaag atataataaa gaatattata tggttaatat agattattta 3360aatagatata tgtatgcgaa ctcacgacaa attgttttta atacacgtag aaataataat 3420gacttcaatg aaggatataa aattataata aaaagaatca gaggaaatac aaatgatact 3480agagtacgag gaggagatat tttatatttt gatatgacaa ttaataacaa agcatataat 3540ttgtttatga agaatgaaac tatgtatgca gataatcata gtactgaaga tatatatgct 3600ataggtttaa gagaacaaac aaaggatata aatgataata ttatatttca aatacaacca 3660atgaataata cttattatta ggcatctcaa atatttaaat caaattttaa tggagaaaat 3720atttctggaa tatgttcaat aggtacttat cgttttagac ttggaggtga ttggtataga 3780cacaattatt tggtgcctac tgtgaagcaa ggaaattatg cttcattatt agaatcaaca 3840tcaactcatt ggggttttgt acctgtaagt gaataa 3876 5 3831 DNA botulinum toxin5 atgacatggc cagtaaaaga ttttaattat agtgatcctg ttaatgacaa tgatatatta 60tatttaagaa taccacaaaa taagttaatt actacacctg taaaagcttt tatgattact 120caaaatattt gggtaatacc agaaagattt tcatcagata ctaatccaag tttaagtaaa 180ccgcccagac ctacttcaaa gtatcaaagt tattatgatc ctagttattt atctactgat 240gaacaaaaag atacattttt aaaagggatt ataaaattat ttaaaagaat taatgaaaga 300gatataggaa aaaaattaat aaattattta gtagttggtt caccttttat gggagattca 360agtacgcctg aagatacatt tgattttaca cgtcatacta ctaatattgc agttgaaaag 420tttgaaaatg gtagttggaa agtaacaaat attataacac caagtgtatt gatatttgga 480ccacttccta atatattaga ctatacagca tcccttacat tgcaaggaca acaatcaaat 540ccatcatttg aagggtttgg aacattatct atactaaaag tagcacctga atttttgtta 600acatttagtg atgtaacatc taatcaaagt tcagctgtat taggcaaatc tatattttgt 660atggatccag taatagcttt aatgcatgag ttaacacatt ctttgcatca attatatgga 720ataaatatac catctgataa aaggattcgt ccacaagtta gcgagggatt tttctctcaa 780gatggaccca acgtacaatt tgaggaatta tatacatttg gaggattaga tgttgaaata 840atacctcaaa ttgaaagatc acaattaaga gaaaaagcat taggtcacta taaagatata 900gcgaaaagac ttaataatat taataaaact attccttcta gttggattag taatatagat 960aaatataaaa aaatattttc tgaaaagtat aattttgata aagataatac aggaaatttt 1020gttgtaaata ttgataaatt caatagctta tattcagact tgactaatgt tatgtcagaa 1080gttgtttatt cttcgcaata taatgttaaa aacaggactc attatttttc aaggcattat 1140ctacctgtat ttgcaaatat attagatgat aatatttata ctataagaga tggttttaat 1200ttaacaaata aaggttttaa tatagaaaat tcgggtcaga atatagaaag gaatcctgca 1260ctacaaaagc ttagttcaga aagtgtagta gatttattta caaaagtatg tttaagatta 1320acaaaaaata gtagagatga ttcaacatgt attaaagtta aaaataatag attaccttat 1380gtagctgata aagatagcat ttcacaagaa atatttgaaa ataaaattat tacagatgag 1440actaatgtac aaaattattc agataatttt tcattagatg aatctatttt agatgggcaa 1500gttcctatta atcctgaaat agtagatcca ctattaccca atgttaatat ggaaccttta 1560aatcttccag gtgaagaaat agtattttat gatgatatta ctaaatatgt tgattattta 1620aattcttatt attatttgga atctcaaaaa ttaagtaata atgttgaaaa tattactctt 1680acaacttcag ttgaagaagc attaggttat agcaataaga tatagacatt tttacctagc 1740ttagctgaaa aagtgaataa aggtgttcaa gcaggtttat tcttaaattg ggcgaatgaa 1800gtagttgagg attttactac aaatattatg aagaaagata cattggataa aatatcagat 1860gtatcagtaa taattccata tataggacct gccttaaata taggaaattc agcattaagg 1920ggaaatttta agcaagcatt tgcaacagct ggtgtagctt ttttattaga gggatttcca 1980gagtttacta tacctgcact cggtgtattt accttttata gttctattca agaaagagag 2040aaaattatta aaactataga aaattgtttg gaacaaagag ttaagagatg gaaagattca 2100tatcaatgga tggtatcaaa ttggttgtca agaattacta ctcaatttaa tcatataaat 2160tatcaaatgt atgattcttt aagttatcag gcagatgcaa tcaaagctaa aatagattta 2220gaatataaaa aatagtcagg aagtgataaa gaaaatataa aaagtcaagt tgaaaattta 2280aaaaatagtt tagatgtaaa aatttcggaa gcaatgaata atataaataa atttatacga 2340gaatgttctg taacatagtt atttaaaaat atgctcccta aagtaattga cgaattaaat 2400aagtttgatt taagaactaa aacagaatta attaatctta tagatagtca taatattatt 2460ctagttggtg aagtagatag attaaaagca aaagtaaatg agagttttga aaatacaatg 2520ccttttaata ttttttcata tactaataat tctttattaa aagatataat taatgaatat 2580ttcaatagta ttaatgattc aaaaattttg agcttacaaa acaaaaaaaa tgctttagtg 2640gatacatcag gatataatgc agaagtgagg gtaggagata atgttcaact taatacgata 2700tatacaaatg actttaaatt aagtagttca ggagataaaa ttatagtaaa tttaaataat 2760aatattttat atagcgctat ttatgagaac tctagtgtta gtttttggat taagatatct 2820aaagatttaa ctaattctca taatgaatat acaataatta acagtataga acaaaattct 2880gggtggaaat tatgtattag gaatggcaat atagaatgga ttttacaaga tgttaataga 2940aagtataaaa gtttaatttt tgattatagt gaatcattaa gtcatacagg atatacaaat 3000aaatggtttt ttgttactat aactaataat ataatggggt atatgaaact ttatataaat 3060ggagaattaa agcagagtca aaaaattgaa gatttagatg aggttaagtt agataaaacc 3120atagtatttg gaatagatga gaatatagat gagaatcaga tgctttggat tagagatttt 3180aatatttttt ctaaagaatt aagtaatgaa gatattaata ttgtatatga gggacaaata 3240ttaagaaatg ttattaaaga ttattgggga aatcctttga agtttgatac agaatattat 3300attattaatg ataattatat agataggtat attgcacctg aaagtaatgt acttgtactt 3360gttcggtatc cagatagatc taaattatat actggaaatc ctattactat taaatcagta 3420tctgataaga atccttatag tagaatttta aatggagata atataattct tcatatgtta 3480tataatagta ggaaatatat gataataaga gatactgata caatatatgc aacacaagga 3540ggagagtgtt cacaaaattg tgtatatgca ttaaaattac agagtaattt aggtaattat 3600ggtataggta tatttagtat aaaaaatatt gtatctaaaa ataaatattg tagtcaaatt 3660ttctctagtt ttagggaaaa tacaatgctt ctagcagata tatataaacc ttggagattt 3720tcttttaaaa atgcatagac gccagttgca gtaactaatt atgaaacaaa actattatca 3780acttcatctt tttggaaatt tatttctagg gatccaggat gggtagagta a 3831 6 3753 DNAbotulinum toxin 6 atgccaacaa ttaatagttt taattataat gatcctgtta ataatagaacaattttatat 60 attaaaccag gcggttgtca acaattttat aaatcattta atattatgaaaaatatttgg 120 ataattccag agagaaatgt aattggtaca attccccaag attttcttccgcctacttca 180 ttgaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtgatcaagaaaag 240 gataaatttt taaaaatagt cacaaaaata tttaatagaa taaatgataatctttcagga 300 aggattttat tagaagaact gtcaaaagct aatccatatt taggaaatgataatactcca 360 gatggtgact tcattattaa tgatgcatca gcagttccaa ttcaattctcaaatggtagc 420 caaagcatac tattacctaa tgttattata atgggagcag agcctgatttatttgaaact 480 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcacggttttggatca 540 atagctatag taacattctc acctgaatat tcttttagat ttaaagataatagtatgaat 600 gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattcattacatgga 660 ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaacaaaatccccta 720 ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggaggtactgattta 780 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagctgattataaa 840 aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaatccttataaa 900 gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaatttattcggtaaat 960 ataaacaaat ttaatgatat ttttaaaaaa ttatacagct ttacggaatttgatttagca 1020 actaaatttc aagttaaatg taggcaaact tatattggac agtataaatacttcaaactt 1080 tcaaacttgt taaatgattc tatttataat atatcagaag gctataatataaataattta 1140 aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattacaccaattaca 1200 ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttctgtaaaaggc 1260 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgtggcttccgag 1320 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatacagtaacttca 1380 aataataatt atgaaaatga tttagatcag gttattttaa attttaatagtgaatcagca 1440 cctggacttt cagatgaaaa attaaattta actatccaaa atgatgcttatataccaaaa 1500 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaacttaatgtattt 1560 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatctcacctcttca 1620 attgatacag cattattaga acaacctaaa atatatacat ttttttcatcagaatttatt 1680 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggatacaacaagtatta 1740 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgcagatatttct 1800 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcacaaaaaggaaat 1860 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttgaacccgagctt 1920 ttaattccta caattttagt attcacgata aaatcttttt taggttcatctgataataaa 1980 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaaatggaaagaa 2040 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatttaataaaaga 2100 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg cacttaaagcaataatagaa 2160 tctaagtata atagttatac tttagaagaa aaaaatgagc ttacaaataaatatgatatt 2220 gagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataatatagacagg 2280 ttcttaactg aaagttctat atcttattta atgaaattaa taaatgaagtaaaaattaat 2340 aaattaagag aatatgatga aaatgttaaa acgtatttat tagattatattataaaacat 2400 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaattgataccctaaat 2460 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaatttcatatttt 2520 aataagttct ttaagagaat taaaagtagt tctgttttaa atatgagatataaaaatgat 2580 aaataggtag atacttcagg atatgattca aatataaata ttaatggagatgtatataaa 2640 tatccaacta ataaaaatca atttggaata tataatgata aacttagtgaagttaatata 2700 tctcaaaatg attacattat atatgataat aaatataaaa attttagtattagtttttgg 2760 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaatacactataata 2820 aattgtatga gggataataa ttcaggatgg aaagtatctc ttaatcataatgaaataatt 2880 tggacattgc aagataattc aggaattaat caaaaattag catttaactatggtaacgca 2940 aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaatgatagatta 3000 ggagattcta aactttatat taatggaaat ttaatagata aaaaatcaattttaaattta 3060 ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtagttatacaaga 3120 tatattggta ttagatattt taatattttt gataaagaat tagatgaaacagaaattcaa 3180 actttatata acaatgaacc taatgcaaat attttaaagg atttttggggaaattatttg 3240 ctttatgaca aagaatagta tttattaaat gtgttaaaac caaataactttattaatagg 3300 agaacagatt ctactttaag cattaataat ataagaagca ctattcttttagctaataga 3360 ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtactaacgataat 3420 cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaactcacttactt 3480 ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaatatcatcatct 3540 ggcaatagat ttaatcaagt agtagttatg aattcagtag gatgtacaatgaattttaaa 3600 aataataatg gaaataatat tgggttgtta ggtttcaagg cagatactgtagttgctagt 3660 acttggtatt atacacatat gagagataat acaaacagca atggatttttttggaacttt 3720 atttctgaag aacatggatg gcaagaaaaa taa 3753 7 3759 DNAbotulinum toxin 7 atgccaaaaa ttaatagttt taattataat gatcctgtta atgatagaacaattttatat 60 attaaaccag gcggttgtca agaattttat aaatcattta atattatgaaaaatatttgg 120 ataattccag agagaaatgt aattggtaca accccccaag attttcatccgcctacttca 180 ttaaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtgatgaagaaaag 240 gatagatttt taaaaatagt cacaaaaata tttaatagaa taaataataatctttcagga 300 gggattttat tagaagaact gtcaaaagct aatccatatt tagggaatgataatactcca 360 gataatcaat tccatattgg tgatgcatca gcagttgaga ttaaattctcaaatggtagc 420 caagacatac tattacctaa tgttattata atgggagcag agcctgatttatttgaaact 480 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcacggttttggatca 540 atagctatag taacattctc acctgaatat tcttttagat ttaatgataatagtatgaat 600 gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattcattacatgga 660 ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaacaaaatccccta 720 ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggaggtactgattta 780 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagctgattataaa 840 aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaatccttataaa 900 gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaatttattcggtaaat 960 ataaacaaat ttaatgatat ttttaaaaaa ttatagagct ttacggaatttgatttagca 1020 actaaatttc aagttaaatg taggcaaact tatattggac agtataaatacttcaaactt 1080 tcaaacttgt taaatgattc tatttataat atatcagaag gctataatataaataattta 1140 aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattacaccaattaca 1200 ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttctgtaaaaggc 1260 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgtggcttccgag 1320 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatacagtaacttca 1380 aataataatt atgaaaatga tttagatcag gttattttaa attttaatagtgaatcagca 1440 cctggacttt cagatgaaaa attaaattta actatccaaa atgatgcttatataccaaaa 1500 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaacttaatgtattt 1560 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatctcacctcttca 1620 attgatacag cattattaga acaacctaaa atatatacat ttttttcatcagaatttatt 1680 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggatacaacaagtgtta 1740 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgcagatatttct 1800 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcacaaaaaggaaat 1860 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttgaacccgagctt 1920 ttaattccta caattttagt attcacgata aaatcttttt taggttcatctgataataaa 1980 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaaatggaaagaa 2040 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatttaataaaaga 2100 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg caattaaaacaataatagaa 2160 tctaagtata atagttatac tttagaggaa aaaaatgagc ttacaaataaatatgatatt 2220 aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataatatagacagg 2280 ttcttaactg aaagttctat atcctattta atgaaattaa taaatgaagtaaaaattaat 2340 aaattaagag aatatgatga gaatgtcaaa acgtatttat tgaattatattatacaacat 2400 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaactgataccctaaat 2460 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaatttcatatttt 2520 aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagatataaaaatgat 2580 aaatacgtag atacttcagg atatgattca aatataaata ttaatggagatgtatataaa 2640 tatccaacta ataaaaatca atttggaata tataatgata aacttagtgaagttaatata 2700 tctcaaaatg attagattat atatgataat aaatataaaa attttagtattagtttttgg 2760 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaatagactataata 2820 aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataatgaaataatt 2880 tggacattgc aagataatgc aggaattaat caaaaattag catttaactatggtaacgca 2940 aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaatgatagatta 3000 ggagattcta aactttatat taatggaaat ttaatagatc aaaaatcaattttaaattta 3060 ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtagttatacaaga 3120 tatattggta ttagatattt taatattttt gataaagaat tagatgaaacagaaattcaa 3180 actttatata gcaatgaacc taatacaaat attttgaagg atttttggggaaattatttg 3240 ctttatgaca aagaatacta tttattaaat gtgttaaaac caaataactttattgatagg 3300 agaaaagatt ctactttaag cattaataat ataagaagca ctattcttttagctaataga 3360 ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtactaacgataat 3420 cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaactcacttattt 3480 ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaatatcatcatct 3540 ggcaatagat ttaatcaagt agtagttatg aattcagtag gaaataattgtacaatgaat 3600 tttaaaaata ataatggaaa taatattggg ttgttaggtt tcaaggcagatactgtagtt 3660 gctagtactt ggtattatac acatatgaga gatcatacaa acagcaatggatgtttttgg 3720 aactttattt ctgaagaaca tggatggcaa gaaaaataa 3759 8 3825DNA botulinum toxin 8 atgccagttg caataaatag ttttaattat aatgaccctgttaatgatga tacaatttta 60 tagatgcaga taccatatga agaaaaaagt aaaaaatattataaagcttt tgagattatg 120 cgtaatgttt ggataattcc tgagagaaat acaataggaacgaatcctag tgattttgat 180 ccaccggctt cattaaagaa cggaagcagt gcttattatgatcctaatta tttaaccact 240 gatgctgaaa aagatagata tttaaaaaca acgataaaattatttaagag aattaatagt 300 aatcctgcag ggaaagtttt gttacaagaa atatcatatgctaaaccata tttaggaaat 360 gaccacacgc caattgatga attctctcca gttactagaactacaagtgt taatataaaa 420 ttatcaacta atgttgaaag ttcaatgtta ttgaatcttcttgtattggg agcaggacct 480 gatatatttg aaagttgttg ttaccccgtt agaaaactaatagatccaga tgtagtttat 540 gatccaagta attatggttt tggatcaatt aatatcgtgacattttcacc tgagtatgaa 600 tatactttta atgatattag tggagggcat aatagtagtacagaatcatt tattgcagat 660 cctgcaattt cactagctca tgaattgata catgcactgcatggattata cggggctagg 720 ggagttactt atgaagagac tatagaagta aagcaagcacctcttatgat agccgaaaaa 780 cccataaggc tagaagaatt tttaaccttt ggaggtcaggatttaaatat tattactagt 840 gctatgaagg aaaaaatata taacaatctt ttagctaactatgaaaaaat agctactaga 900 cttagtgaag ttaatagtgc tcctcctgaa tatgatattaatgaatataa agattatttt 960 caatggaagt atgggctaga taaaaatgct gatggaagttatactgtaaa tgaaaataaa 1020 tttaatgaaa tttataaaaa attatatagt tttacagagagtgacttagc aaataaattt 1080 aaagtaaaat gtagaaatac ttattttatt aaatatgaatttttaaaagt tccaaatttg 1140 ttagatgatg atatttatac tgtatcagag gggtttaatataggtaattt agcagtaaac 1200 aatcgcggac aaagtataaa gttaaatcct aaaattattgattccattcc agataaaggt 1260 ctagtagaaa agatcgttaa attttgtaag agcgttattcctagaaaagg tacaaaggcg 1320 ccaccgcgac tatgcattag agtaaataat agtgagttattttttgtagc ttcagaaagt 1380 agctataatg aaaatgatat taatacacct aaagaaattgacgatacaac aaatctaaat 1440 aataattata gaaataattt agatgaagtt attttagattataatagtca gacaatacct 1500 caaatatcaa atcgaacatt aaatacactt gtacaagacaatagttatgt gccaagatat 1560 gattctaatg gaacaagtga aatagaggaa tatgatgttgttgactttaa tgtatttttc 1620 tatttacatg cacaaaaagt gccagaaggt gaaaccaatataagtttaac ttcttcaatt 1680 gatacagcat tattagaaga atccaaagat atatttttttcttcagagtt tatcgatact 1740 atcaataaac ctgtaaatgc agcactattt atagattggataagcaaagt aataagagat 1800 tttaccactg aagctacaca aaaaagtact gttgataagattgcagacat atctttaatt 1860 gtaccctatg taggtcttgc tttgaatata attattgaggcagaaaaagg aaattttgag 1920 gaggcatttg aattattagg agtgggtatt ttattagaatttgtgccaga acttacaatt 1980 cctgtaattt tagtgtttac gataaaatcc tatatagattcatatgagaa taaaaataaa 2040 gcaattaaag caataaataa ttcattaatc gaaagagaagcaaagtggaa agaaatatat 2100 agttggatag tatcaaattg gcttactaga attaatactcaatttaataa aagaaaagag 2160 caaatgtatc aggctttaca aaatcaagta gatgcaataaaaacagcaat agaatataaa 2220 tataataatt atacttcaga tgagaaaaat agacttgaatctgaatataa tatcaataat 2280 atagaagaag aattgaataa aaaagtttct ttagcaatgaaaaatataga aagatttatg 2340 acagaaagtt ctatatctta tttaatgaaa ttaataaatgaagccaaagt tggtaaatta 2400 aaaaaatatg ataaccatgt taagagcgat ttattaaactatattctcga ccatagatca 2460 atcttaggag agcagacaaa tgaattaagt gatttggtgactagtacttt gaatagtagt 2520 attccatttg aactttcttc atatactaat gataaaattctaattatata ttttaataga 2580 ttatataaaa aaattaaaga tagttctatt ttagatatgcgatatgaaaa taataaattt 2640 atagatatct ctggatatgg ttcaaatata agcattaatggaaacgtata tatttattca 2700 acaaatagaa atcaatttgg aatatataat agtaggcttagtgaagttaa tatagctcaa 2760 aataatgata ttatatagaa tagtagatat caaaattttagtattagttt ctgggtaagg 2820 attcctaaac actagaaacc tatgaatcat aatcgggaatagactataat aaattgtatg 2880 gggaataata attcgggatg gaaaatatca cttagaactgttagagattg tgaaataatt 2940 tggactttac aagatacttc tggaaataag gaaaatttaatttttaggta tgaagaactt 3000 aataggatat ctaattatat aaataaatgg atttttgtaactattactaa taatagatta 3060 ggcaattcta gaatttagat caatggaaat ttaatagttgaaaaatcaat ttcgaattta 3120 ggtgatattc atgttagtga taatatatta tttaaaattgttggttgtga tgatgaaacg 3180 tatgttggta taagatattt taaagttttt aatacggaattagataaaac agaaattgag 3240 actttatata gtaatgagcc agatccaagt atcttaaaaaactattgggg aaattatttg 3300 ctatataata aaaaatatta tttattcaat ttactaagaaaagataagta tattactctg 3360 aattcaggca ttttaaatat taatcaacaa agaggtgttactgaaggctc tgtttttttg 3420 aactataaat tatatgaagg agtagaagtc attataagaaaaaatggtcc tatagatata 3480 tctaatacag ataattttgt tagaaaaaac gatctagcatacattaatgt agtagatcgt 3540 ggtgtagaat atcggttata tgctgataca aaatcagagaaagagaaaat aataagaaca 3600 tctaatctaa acgatagctt aggtcaaatt atagttatggattcaatagg aaataattgc 3660 acaatgaatt ttcaaaacaa taatgggagc aatataggattactaggttt tcattcaaat 3720 aatttggttg ctagtagttg gtattataac aatatacgaagaaatactag cagtaatgga 3780 tgcttttgga gttctatttc taaagagaat ggatggaaagaatga 3825 9 3894 DNA Artificial Sequence Description of ArtificialSequence synthetic primers used to introduce Stu I and EcoR Irestriction sites into the 5′ and 3′ ends of the BoNT/A-L chain genefragment 9 atgccagtta atataaaaaa ctttaattat aatgacccta ttaataatgatgacattatt 60 atgatggaac cattcaatga cccagggcca ggaacatatt ataaagcttttaggattata 120 gatcgtattt ggatagtacc agaaaggttt acttatggat ttcaacctgaccaatttaat 180 gccagtacag gagtttttag taaagatgtc tacgaatatt aggatccaacttatttaaaa 240 accgatgctg aaaaagataa atttttaaaa acaatgatta aattatttaatagaattaat 300 tcaaaaccat caggacagag attactggat atgatagtag atgctataccttatcttgga 360 aatgcatcta caccgcccga caaatttgca gcaaatgttg caaatgtatctattaataaa 420 aaaattatcc aacctggagc tgaagatcaa ataaaaggtt taatgacaaatttaataata 480 tttggaccag gaccagttct aagtgataat tttactgata gtatgattatgaatggccat 540 tccccaatat cagaaggatt tggtgcaaga atgatgataa gattttgtcctagttgttta 600 aatgtattta ataatgttca ggaaaataaa gatacatcta tatttagtagacgcgcgtat 660 tttgcagatc cagctctaac gttaatgcat gaacttatac atgtgttacatggattatat 720 ggaattaaga taagtaattt accaattact ccaaatacaa aagaatttttcatgcaacat 780 agcgatcctg tacaagcaga agaactatat acattcggag gacatgatcctagtgttata 840 agtccttcta cggatatgaa tatttataat aaagcgttac aaaattttcaagatatagct 900 aataggctta atattgtttc aagtgcccaa gggagtggaa ttgatatttccttatataaa 960 caaatatata aaaataaata tgattttgtt gaagatccta atggaaaatatagtgtagat 1020 aaggataagt ttgataaatt atataaggcc ttaatgtttg gctttactgaaactaatcta 1080 gctggtgaat atggaataaa aactaggtat tcttatttta gtgaatatttgccaccgata 1140 aaaactgaaa aattgttaga caatacaatt tatactcaaa atgaaggctttaacatagct 1200 agtaaaaatc tcaaaacgga atttaatggt cagaataagg cggtaaataaagaggcttat 1260 gaagaaatca gcctagaaca tctcgttata tatagaatag caatgtgcaagcctgtaatg 1320 tacaaaaata ccggtaaatc tgaacagtgt attattgtta ataatgaggatttatttttc 1380 atagctaata aagatagttt ttcaaaagat ttagctaaag cagaaactatagcatataat 1440 acacaaaata atactataga aaataatttt tctatagatc agttgattttagataatgat 1500 ttaagcagtg gcatagactt accaaatgaa aacacagaac catttacaaattttgacgac 1560 atagatatcc ctgtgtatat taaacaatct gctttaaaaa aaatttttgtggatggagat 1620 agcctttttg aatatttaca tgctcaaaca tttccttcta atatagaaaatctacaacta 1680 acgaattcat taaatgatgc tttaagaaat aataataaag tctatacttttttttctaca 1740 aaccttgttg aaaaagctaa tacagttgta ggtgcttcac tttttgtaaactgggtaaaa 1800 ggagtaatag atgattttac atctgaatcc acacaaaaaa gtactatagataaagtttca 1860 gatgtatcca taattattcc ctatatagga cctgctttga atgtaggaaatgaaacagct 1920 aaagaaaatt ttaaaaatgc ttttgaaata ggtggagccg ctatcttaatggagtttatt 1980 ccagaactta ttgtacctat agttggattt tttacattag aatcatatgtaggaaataaa 2040 gggcatatta ttatgacgat atccaatgct ttaaagaaaa gggatcaaaaatggacagat 2100 atgtatggtt tgatagtatc gcagtggctc tcaacggtta atactcaattttatacaata 2160 aaagaaagaa tgtagaatgc tttaaataat caatcacaag caatagaaaaaataatagaa 2220 gatcaatata atagatatag tgaagaagat aaaatgaata ttaacattgattttaatgat 2280 atagatttta aacttaatca aagtataaat ttagcaataa acaatatagatgattttata 2340 aaccaatgtt ctatatcata tctaatgaat agaatgattc cattagctgtaaaaaagtta 2400 aaagactttg atgataatct taagagagat ttattggagt atatagatacaaatgaacta 2460 tatttacttg atgaagtaaa tattctaaaa tcaaaagtaa atagacacctaaaagacagt 2520 ataccatttg atctttcact atataccaag gacacaattt taatacaagtttttaataat 2580 tatattagta atattagtag taatgctatt ttaagtttaa gttatagaggtgggcgttta 2640 atagattcat ctggatatgg tgcaactatg aatgtaggtt cagatgttatctttaatgat 2700 ataggaaatg gtcaatttaa attaaataat tctgaaaata gtaatattacggcacatcaa 2760 agtaaattcg ttgtatatga tagtatgttt gataatttta gcattaacttttgggtaagg 2820 actcctaaat ataataataa tgatatacaa acttatcttc aaaatgagtatacaataatt 2880 agttgtataa aaaatgactc aggatggaaa gtatctatta agggaaatagaataatatgg 2940 acattaatag atgttaatgc aaaatctaaa tcaatatttt tcgaatatagtataaaagat 3000 aatatatcag attatataaa taaatggttt tccataacta ttactaatgatagattaggt 3060 aacgcaaata tttatataaa tggaagtttg aaaaaaagtg aaaaaattttaaacttagat 3120 agaattaatt ctagtaatga tatagacttc aaattaatta attgtacagatactactaaa 3180 tttgtttgga ttaaggattt taatattttt ggtagagaat taaatgctacagaagtatct 3240 tcactatatt ggattcaatc atctacaaat actttaaaag atttttgggggaatccttta 3300 agataggata cacaatacta tctgtttaat caaggtatgc aaaatatctatataaagtat 3360 tttagtaaag cttctatggg ggaaactgca ccacgtacaa actttaataatgcagcaata 3420 aattatcaaa atttatatct tggtttacga tttattataa aaaaagcatcaaattctcgg 3480 aatataaata atgataatat agtcagagaa ggagattata tatatcttaatattgataat 3540 atttctgatg aatcttagag agtatatgtt ttggtgaatt ctaaagaaattcaaactcaa 3600 ttatttttag cacccataaa tgatgatcct acgttctatg atgtactacaaataaaaaaa 3660 tattatgaaa aaacaacata taattgtcag atactttgcg aaaaagatactaaaacattt 3720 gggctgtttg gaattggtaa atttgttaaa gattatggat atgtttgggatacctatgat 3780 aattattttt gcataagtca gtggtatctc agaagaatat ctgaaaatataaataaatta 3840 aggttgggat gtaattggca attcattccc gtggatgaag gatggacagaataa 3894 10 23 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide used to introduce Stu I and EcoR I intoBoNT/A-L chain gene fragments 10 aaaggccttt tgttaataaa caa 23 11 26 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideused to introduce Stu I and EcoR I into BoNT/A-L chain gene fragment 11ggaattctta cttattgtat ccttta 26 12 13 PRT Artificial SequenceDescription of Artificial Sequence polypeptide fragment used to raiseantibodies 12 Cys Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10

What is claimed is:
 1. A method for treating hyperhidrosis in a mammal,said method comprising the step of locally administering a drug particleto an affected skin area without using a needle.
 2. The method of claim1 wherein the skin comprises an epidermis layer, a dermis layer and ahypodermis layer.
 3. The method of claim 1 wherein the drug particle isadministered to a dermis layer of the skin.
 4. The method of claim 1wherein the drug particle is administered to one or more layers of theskin where a sweat gland and/or a nerve innervating a sweat gland islocated.
 5. The method of claim 1 wherein the drug particle isadministered to the skin and substantially not to a muscle tissue. 6.The method of claim 1 wherein the drug particle is administered to adermis layer of the skin and substantially not to a muscle tissue. 7.The method of claim 1 wherein the step of administering includes using aneedleless injector.
 8. The method of claim 7 wherein the drug particleis administered to a dermis layer of a skin and substantially not to amuscle tissue.
 9. The method of claim 1 wherein the drug particlecomprises a neurotoxin.
 10. The method of claim 9 wherein the neurotoxincomprises: (a) a targeting component; (b) a therapeutic component; and(c) a translocation component.
 11. The method of claim 10 wherein thetargeting component binds to a presynaptic nerve terminal.
 12. Themethod of claim 12 wherein the presynaptic nerve terminal belongs to acholinergic neuron.
 13. The method of claim 11 wherein the targetingcomponent comprises a carboxyl end segment of a heavy chain of abutyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E,F, G or a variant thereof.
 14. The method of claim 11 wherein thetargeting component comprises a carboxyl end fragment of a heavy chainof a botulinum toxin type A.
 15. The method of claim 11 wherein thetherapeutic component substantially interferes with the release ofneurotransmitters from a neuron or its terminals.
 16. The method ofclaim 11 wherein the therapeutic component comprises a light chain of abutyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E,F, G or a variant thereof.
 17. The method of claim 11 wherein thetherapeutic component comprises a light chain of a botulinum toxin typeA.
 18. The method of claim 11 wherein the translocation componentfacilitates the transfer of at least a part of the neurotoxin into thecytoplasm of the target cell.
 19. The method of claim 11 wherein thetranslocation component comprises an amino end fragment of a heavy chainof a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁,D, E, F, G or a variant thereof.
 20. The method of claim 11 wherein thetranslocation component comprises an amino end fragment of a heavy chainof a botulinum toxin type A.
 21. The method of claim 11 wherein thetargeting component comprises a carboxyl end fragment of a heavy chainof a botulinum toxin type A, the therapeutic component comprises a lightchain of a botulinum toxin type A and the translocation componentcomprises an amine end fragment of a heavy chain of a botulinum toxintype A.
 22. The method of claim 11 wherein the neurotoxin is botulinumtoxin type A.
 23. The method of claim 1 wherein the neurotoxin isrecombinantly produced.
 24. The method of claim 1 wherein the drugparticle comprises a neurotoxin and a carrier, wherein the neurotoxin iscoated onto the carrier.
 25. The method of claim 24 wherein the carrieris a dense material selected from the group consisting of gold, platinumand ice crystal.
 26. The method of claim 9 wherein the neurotoxincomprises a nucleotide sequence.
 27. The method of claim 26 wherein thenucleotide sequence is SEQ. ID. #1, variants thereof or fragmentsthereof.
 28. The method of claim 26 wherein the nucleotide sequence isselected from the group consisting of SEQ. ID #2, SEQ. ID. #3, SEQ. ID.#4, SEQ. ID. #5, SEQ. ID. #6, SEQ. ID. #7, SEQ. ID. #8 and SEQ. ID. #9,variants thereof or fragments thereof.
 29. A method for treatinghyperhidrosis in a mammal, said method comprising the step of using aneedleless injector to locally administer a drug particle comprising aneurotoxin to a dermis layer of an affected area of a skin.
 30. Themethod of claim 29 wherein the neurotoxin comprises botulinum toxin typeA.